Phase IIa Randomized, Single-Blinded, Placebo-Controlled Clinical Trial of the Reprofiled Drug Auranofin for GI Protozoa
Phase: PHASE2
Conditions: Amoebic Dysentery, Giardiasis
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)

________________________________________________________________________________________________________ 
 
 
Phase IIa Randomized, Single- blinded, Placebo-controlled Clinical 
Trial of the Reprofiled Drug 
Auranofin for GI Protozoa 
 
DMID Protocol Number: 15-0015 
 
 
DMID Funding Mechanism: U01AI110435-01 
 
 
Principal Investigator: Sharon Reed, MD 
 
 
DMID Program Officer: Greg Deye, MD 
 
 
DMID Medical Monitor: Mo Elsafy, MD 
 
 
Version Number: 8.0 
 
 
14 Apr 2020 
 
 
 
 
 
 

DMID Protocol 15-0015 
Version 8.0                                              14 APR 2020 
 
 
________________________________________________________________________________________________________ 
i 
Statement of Compliance 
This trial will be conducted in accordance with Good Clinical Practice (GCP) as required by the 
following: 
• United States (US) Code of Federal Regulations (CFR) applicable to clinical studies 
(45 CFR Part 46; 21 CFR Part 50, 21 CFR Part 56, and 21 CFR Part 312) 
ICH E6; 62 Federal Register 25691 (1997)  
• NIH Clinical Terms of Award 
 
All key personnel (all individuals responsible for the design and conduct of this study) have 
completed Human Subjects Protection Training. 

DMID Protocol 15-0015 
Version 8.0                                              14 APR 2020 
 
 
________________________________________________________________________________________________________ 
ii 
Signature Page 
The signature below constitutes the approval of this protocol and the attachments, and provides 
the necessary assurances that this trial will be conducted according to all stipulations of the 
protocol, including all statements regarding confidentiality, and according to local legal and 
regulatory requirements and applicable U.S. federal regulations and ICH guidelines. 
 
Site Investigator:* 
 
Signed: 
 
Date: 
 
 
Sharon Reed, MD 
 
 
 
*The protocol should be signed by the local investigator who is responsible for the study 
implementation at his/her specific site; i.e., if Investigational New Drug study, the individual 
who signs the Form FDA 1572. 
 

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iii 
TABLE OF CONTENTS 
 
  
PAGE 
STATEMENT OF COMPLIANCE ................................................................................................. I 
SIGNATURE PAGE ...................................................................................................................... II 
TABLE OF CONTENTS .............................................................................................................. III 
LIST OF FIGURES ..................................................................................................................... VII 
LIST OF ABBREVIATIONS .................................................................................................... VIII 
PROTOCOL SUMMARY .............................................................................................................. 1 
SCHEMATIC OF STUDY DESIGN: ............................................................................................ 4 
1 
KEY ROLES ........................................................................................................................... 6 
2 
BACKGROUND INFORMATION AND SCIENTIFIC RATIONALE ................................ 9 
2.1 
Background Information .................................................................................................. 9 
2.1.1 Summary of Previous Pre-clinical Studies ................................................................... 9 
2.1.2 Summary of Relevant Clinical Studies ....................................................................... 12 
2.1.3 Summary of Epidemiological Data ............................................................................ 13 
2.2 
Rationale ......................................................................................................................... 13 
2.3 
Potential Risks and Benefits ........................................................................................... 14 
2.3.1 Potential Risks ............................................................................................................ 14 
2.3.2 Known Potential Benefits ............................................................................................. 15 
3 
OBJECTIVES ........................................................................................................................ 16 
3.1 Study Objectives ................................................................................................................. 16 
3.2 
Study Outcome Measures............................................................................................... 17 
3.2.1 Primary Outcome Measures ......................................................................................... 17 
3.2.2 Secondary Outcome Measures ..................................................................................... 18 
4 
STUDY DESIGN .................................................................................................................. 19 
4.1 
Sub Studies ..................................................................................................................... 19 
5 
STUDY ENROLLMENT AND WITHDRAWAL ............................................................... 20 
5.1 
Subject Inclusion Criteria ............................................................................................... 20 
5.2 
Subject Exclusion Criteria .............................................................................................. 22 
5.3 
Treatment Assignment Procedures ................................................................................. 23 
5.3.1 Enrollment and Randomization Procedures ............................................................... 23 
5.3.2 Masking Procedures ................................................................................................... 24 
5.3.3 Reasons for Withdrawal ............................................................................................. 24 

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5.3.4 Handling of Withdrawals............................................................................................ 25 
5.3.5 Termination of Study .................................................................................................. 26 
6 
STUDY INTERVENTION/INVESTIGATIONAL PRODUCT .......................................... 27 
6.1 
Study Product Description ............................................................................................. 27 
6.1.1 Acquisition.................................................................................................................. 27 
6.1.2 Formulation, Packaging, and Labeling ....................................................................... 27 
6.2 
Dosage, Preparation and Administration of Study Intervention/Investigational Product
........................................................................................................................................ 28 
6.3 
Modification of Study Intervention/Investigational Product for a Participant ............... 29 
6.4 
Accountability Procedures for the Study Intervention/Investigational Product(s) ........ 29 
6.5 
Assessment of Subject Compliance with Study Intervention/Investigational Product(s)
........................................................................................................................................ 29 
6.6 
Concomitant Medications/Treatments ........................................................................... 29 
7 
STUDY SCHEDULE ............................................................................................................ 31 
7.1 
Screening Visits .............................................................................................................. 31 
7.1.1 Visit 00A, Day -4 to -1, Screening 1, icddr,b or Rajshahi Medical College Hospitals
 ................................................................................................................................... 31 
7.1.2 Visit 00B, Day -4 to -1, Screening 2, icddr, b or  Rajshahi Medical College Hospitals
 ................................................................................................................................... 31 
7.2 
Visit 01, Day 1, Enrollment, Dose 1, icddr,b or Rajshahi Medical College Hospital or 
Clinic .............................................................................................................................. 32 
7.3 
Follow-up Visits ............................................................................................................. 33 
7.3.1 Visit 02, Day 2 ............................................................................................................ 33 
7.3.2 Visit 03, Day 3 ............................................................................................................ 33 
7.3.3 Visit 04, Day 4 ............................................................................................................ 34 
7.3.4 Visit 05, Day 5 ............................................................................................................ 34 
7.3.5 Visit 06, Day 6 (Amebiasis arm only) ........................................................................ 34 
7.3.6 Visit 07, Day 7 (Window + 1 day) icddr,b or Rajshahi Medical College Hospitals .. 35 
7.3.7 Visit 08, Day 14 (Window: +/- 3 days) ........................................................................ 35 
7.3.8 Visit 09, Day 28 (Window: +/- 3 days) ...................................................................... 36 
7.3.9 Visit 10, Day 30 (Window: +/- 3 days) ...................................................................... 36 
7.4 
Early Termination Visit .................................................................................................. 36 
7.5 
Unscheduled Visit .......................................................................................................... 36 
7.6 
Pregnancy Visit .............................................................................................................. 37 
8 
STUDY PROCEDURES/EVALUATIONS.......................................................................... 38 
8.1 
Clinical Evaluations ....................................................................................................... 38 
8.2 Laboratory Evaluations ....................................................................................................... 38 
8.2.1 Clinical Laboratory Evaluations ................................................................................... 38 
8.2.2 Specimen Preparation, Handling and Shipping ............................................................ 40 

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9 
ASSESSMENT OF SAFETY ............................................................................................... 41 
9.1 Specification of Safety Parameters ..................................................................................... 41 
9.1 
42 
9.2 
Methods and Timing for Assessing, Recording, and Analyzing Safety Parameters ...... 42 
9.2.1 Adverse Events ............................................................................................................. 42 
9.2.2 Solicited Events ............................................................................................................ 43 
9.2.3 Additional Adverse Event Severity Grading ................................................................ 45 
9.2.4 Serious Adverse Events ................................................................................................ 47 
9.2.5 Procedures to be Followed in the Event of Abnormal Laboratory Test Values or 
Abnormal Clinical Findings ...................................................................................... 47 
9.3 Reporting Procedures .......................................................................................................... 48 
9.3.1 Serious Adverse Events ................................................................................................ 48 
9.3.2 Regulatory Reporting for Studies Conducted Under DMID-Sponsored IND .............. 49 
9.3.3 Reporting of Pregnancy ................................................................................................ 49 
9.4 Type and Duration of the Follow-up of Participants after Adverse Events ........................ 49 
9.4.1. Halting Rules .................................................................................................................. 50 
9.4.2. Safety Oversight (DSMB) .............................................................................................. 51 
9.4.2 Independent Safety Monitor (ISM) ............................................................................ 51 
9.4.3 Data Safety Monitoring Board (DSMB) .................................................................... 51 
10. CLINICAL MONITORING STRUCTURE ......................................................................... 53 
10.1 Site Monitoring Plan ......................................................................................................... 53 
11. STATISTICAL CONSIDERATIONS .................................................................................. 54 
11.1 Study Hypotheses .............................................................................................................. 54 
11.2 Sample Size Considerations .............................................................................................. 54 
11.3 Planned Interim Analyses .................................................................................................. 55 
11.3.1 Safety Review ............................................................................................................. 55 
11.4 Final Analysis Plan............................................................................................................ 56 
12 SOURCE DOCUMENTS AND ACCESS TO SOURCE DATA/DOCUMENTS .............. 59 
13 QUALITY CONTROL AND QUALITY ASSURANCE .................................................... 60 
14 ETHICS/PROTECTION OF HUMAN SUBJECTS ............................................................... 61 
14.1 Ethical Standard ................................................................................................................ 61 
14.2 Institutional Review Board/Ethics Committee .................................................................. 61 
14.3 Informed Consent Process ................................................................................................. 61 
14.4 Exclusion of Women, Minorities, and Children  (Special Populations) ........................... 63 
14.5 Subject Confidentiality ...................................................................................................... 63 
14.6 Study Discontinuation ....................................................................................................... 64 
14.7 Future Use of Stored Specimens ....................................................................................... 64 
15 DATA HANDLING AND RECORD KEEPING ................................................................... 65 

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15.1 Data Management Responsibilities ................................................................................... 65 
15.2 Data Capture Methods ....................................................................................................... 65 
15.3 Types of Data .................................................................................................................... 66 
15.4 Timing/Reports.................................................................................................................. 66 
15.5 Study Records Retention ................................................................................................... 66 
15.6 Protocol Deviations ........................................................................................................... 66 
16 PUBLICATION POLICY ....................................................................................................... 68 
17 SCIENTIFIC REFERENCES .................................................................................................. 70 
APPENDIX A:   SCHEDULE OF EVENTS ................................................................................. 1 
APPENDIX B:LAB PROCESSING FLOW SHEET: CLINICAL/ SAFETY LAB TESTS ......... 4 
APPENDIX C: ICDDR,B NORMAL LABORATORY VALUES (APPLICABLE FOR BOTH 
SITES) ................................................................................................................................... 6 
 
 
 

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LIST OF FIGURES 
Figure 1: Killing of metronidazole-sensitive and resistant strains by metronidazole (Mz) and 
auranofin (AF) .............................................................................................................................. 10 
Figure 2: Effect of auranofin on amebic colitis in mice. .............................................................. 10 
Figure 3: Efficacy of auranofin in rodent models of giardiasis .................................................... 11 
Figure 4: Efficacy of auranofin and metronidazole against Entamoeba cysts .............................. 12 
 
 

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List of Abbreviations 
AE 
Adverse Event/Adverse Experience 
CAR 
Clinical Agents Repository 
CFR 
Code of Federal Regulations 
CIOMS 
Council for International Organizations of Medical Sciences 
CONSORT 
Consolidated Standards of Reporting Trials 
CRF 
Case Report Form 
CRO 
Contract Research Organization 
DHHS 
Department of Health and Human Services 
DMID 
Division of Microbiology and Infectious Diseases, NIAID, NIH, 
DHHS 
DSMB 
Data and Safety Monitoring Board 
eCRF 
Electronic Case Report Form 
FDA 
Food and Drug Administration 
FWA 
Federalwide Assurance 
GCP 
Good Clinical Practice 
HIPAA 
Health Insurance Portability and Accountability Act 
IB 
Investigator’s Brochure 
icddr,b 
International Centre for Diarrhoeal Disease Research-
Bangladesh 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
ICMJE 
International Committee of Medical Journal Editors 
IDE 
Investigational Device Exemption 
IEC 
Independent or Institutional Ethics Committee 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
JAMA 
Journal of the American Medical Association 
MedDRA 
Medical Dictionary for Regulatory Activities 
MOP 
Manual of Procedures 
N 
Number (typically refers to subjects) 
NCI 
National Cancer Institute, NIH, DHHS 
NDA 
New Drug Application 
NEJM 
New England Journal of Medicine 
NIAID 
National Institute of Allergy and Infectious Diseases, NIH, 
DHHS 

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NIH 
National Institutes of Health 
OCRA 
Office of Clinical Research Affairs, DMID, NIAID, NIH, 
DHHS 
OHRP 
Office for Human Research Protections 
OHSR 
Office for Human Subjects Research 
ORA 
Office of Regulatory Affairs, DMID, NIAID, NIH, DHHS 
PHI 
Protected Health Information 
PI 
Principal Investigator 
PK 
Pharmacokinetics 
QA 
Quality Assurance 
QC 
Quality Control 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SDCC 
Statistical and Data Coordinating Center 
SMC 
Safety Monitoring Committee 
SOP 
Standard Operating Procedure 
US 
United States 
WHO 
World Health Organization 
 
 

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1 
Protocol Summary 
Title: 
Phase IIa Randomized, Single-blinded, Placebo-controlled 
Clinical Trial of the Reprofiled Drug, Auranofin for GI Protozoa 
Short Title: 
Auranofin for GI Protozoa 
Phase: 
IIa 
Population: 
136 symptomatic adults >18 to 65 years old from Bangladesh with 
amebiasis or giardiasis 
Number of Sites: 
1 
Study Duration:  
3.5 years 
Subject Participation 
Duration: 
7 days treatment for amebiasis and 5 days for giardiasis with follow-
up through Day 28 
Description of Agent of 
Intervention: 
Auranofin (Ridaura) 6 mg, or similar placebo 
Objectives: 
Primary E. histolytica: 
• To compare the proportion of subjects with stools positive by 
rapid EIA and positive antigen detection EIA for E. histolytica at 
enrollment with resolution of diarrhea (less than 3 loose stools/24 
hrs) by Day 7  
Secondary E. histolytica: 
• To compare the proportion of subjects with stools positive by 
rapid EIA and positive antigen detection EIA for E. histolytica 
and trophozoites on smear at enrollment with parasitological 
response (no detection of trophozoites of E. histolytica on 
microscopic exam by Days 7 
• To compare the proportion of subjects with stools positive rapid 
EIA and positive antigen detection EIA for E. histolytica and 
trophozoites on smear at enrollment with parasitological response 

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(no detection of trophozoites on microscopic exam or negative 
antigen detection) by Day 3 and 5 
• To compare the rate of decrease of trophozoites/cyst load by 
qPCR in stools by Days 3, 5, and 7 
• To compare the proportion of subjects with negative stool antigen 
tests by days 3, 5, 7, and 14 
• To compare the proportion of subjects with sustained cure (no 
detection of trophozoites by microscopic exam) at 14 and 28 days  
• To compare the proportion of subjects with relapse (same strain) 
or re-infection (new strain) with positive stools at 14 and 28 days 
by genotyping the initial vs. subsequent strain 
• To compare the time to resolution of diarrhea (less than 3 loose 
stools/24 hours) 
Primary Giardia: 
• To compare the proportion of subjects with stools positive by 
rapid EIA and positive antigen detection EIA for Giardia at 
enrollment with resolution of diarrhea (less than 3 loose stools/24 
hours) by Day 5  
Secondary Giardia: 
• To compare the proportion of subjects with parasitological 
response (no detection of trophozoites on microscopic exam) on 
Days 3 and 5  
 
• To compare the rate of decrease of trophozoites/cyst load by qPCR 
in stools by Days 3 and 5 
 
• To compare the proportion of subjects with negative stool antigens 
by days 3 and 5. 
• To compare the proportion of subjects with sustained cure (no 
detection of r trophozoites by microscopic exam)  at 14 and 28 
days  

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• To compare the proportion of subjects with relapse (same strain) 
or re-infection (new strain) with positive stools at 14 and 28 days 
by genotyping the initial vs. subsequent strain 
• To compare the time to resolution of diarrhea (less than 3 loose 
stools/24 hours) 
Description of Study 
Design: 
Randomized, placebo-controlled, single-blinded superiority treatment 
study to compare placebo to once daily doses of auranofin for adults 
with amebiasis or giardiasis. A sample size of 68 subjects enrolled 
with amebiasis (34 per arm) and 68 with giardiasis (34 per arm); 
Power based on 60 subjects with amebiasis and 60 with giardiasis 
completing the study 
Estimated Time to 
Complete Enrollment: 
3.5 years 

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Schematic of Study Design: 
 
 
Prior to Enrollment 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Day 1 
 
 
 
Day 2 
 
 
 
Day 3 
 
 
 
 
Day 4 
 
N=34 
Arm 1 
Active
Clinical Assessment, Obtain stool specimen 
Administer Study Drug 
N=68 Positive for E. histolytica 
 
N=68 Positive for G. lamblia 
Clinical and AE Assessment 
Administer Study Drug 
N=34 
Arm 1 
Active 
N=34 
Arm 2 
Placebo 
N=34 
Arm 2 
Placebo 
Clinical and AE Assessment 
Administer Study Drug 
Clinical and AE Assessment, Obtain stool specimen 
Administer Study Drug 
Obtain informed consent. Screen subjects by 
criteria. Obtain demographic/clinical data, and stool specimen at visit 00A 
and screening blood, urine, and pregnancy testing at Visit 00B.   

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5 
 
 
 
Day 5 
 
 
 
 
Day 6 
 
 
 
Day 7 
 
 
 
 
Day 14 
 
 
 
 
 
Day 28 
 
 
 
 
 
 
 
 
  
Day 30 
Clinical and AE Assessment, Obtain urine, blood 
Administer Study Drug and obtain stool for Amebiasis patients only 
Clinical and AE Assessment 
Administer Study Drug to Amebiasis patients only 
Clinical and AE Assessment 
Obtain stool specimen 
Clinical and AE Assessment, Obtain stool specimen 
Discharge from Study 
Administer standard of care treatment with metronidazole to all 
subjects who are still positive at Day 28 for Giardia or for E. 
histolytica
 
 
Clinical and AE Assessment, Obtain stool specimen 
Administer Study Drug 

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1 
KEY ROLES 
Institutions: University of California, San Diego 
 
9500 Gilman Dr. 
 
La Jolla, CA 92093-0612 
 
Contact Person: Sharon Reed, MD 
 
Phone: 858-822-2808 
 
Fax: 858-534-5724 
 
Email: slreed@ucsd.edu 
 
 
 
University of Virginia School of Medicine 
 
 
Carter Harrison Building MR6, Rm. 1709 
PO Box 801340 
345 CRISPELL DR 
 
Charlottesville, VA 22908  
 
Contact Person: William Petri, Jr., MD, PhD 
 
Phone: 434-924-5621 
 
Fax: 434-924-0075 
 
Email: wap3g@virginia.edu 
 
 
International Center for Diarrheal Disease Research, Bangladesh 
 
Mohakhali, Dhaka-1212 
 
Bangladesh 
 
Contact Person: Dr. Rashidul Haque, MD, PhD 
 
Phone: 880-2-8860523 
 
Fax: 880 28812529 
 
Email: rhaque@icddrb.org 
 
 
Rajshahi Medical College Hospital 
 
Rajshahi 6000, Bangladesh 
 
Contact Person: Dr. Faisal Alam, MD 
 
Phone cell: +8801558368966 
 
Email: faisal_imm@yahoo.com 
 
 
 

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7 
 
 
INDIVIDUALS: 
 
Principal Investigator: 
 
 
 
 
Program Officer/Medical Officer: 
 
 
Sharon Reed, MD 
University of California, San Diego 
9500 Gilman Dr. 
La Jolla, CA 92093-0612 
Contact Person: Sharon Reed, MD 
Phone: 858-822-2808 
Fax: 858-534-5724 
Email: slreed@ucsd.edu 
 
Gregory A. Deye, MD 
Parasitology & International Programs Branch (HNM53) 
Division of Microbiology & Infectious Diseases 
National Institute of Allergy & Infectious Diseases 
National Institutes of Health 
BG 5601FL RM 8A39 
Mail Stop 9825 
5601 Fishers Lane 
Bethesda, MD 20852 
 
Medical Monitor: 
 
 
 
 
 
 
Mo Elsafy, MD,  
MSc Medical Monitor Office of Clinical Research Affairs  
Division of Microbiology and Infectious Diseases  
5601 Fishers Lane 7E56 MSC 9826 Bethesda, MD 20892 
Mail Stop: 9826  
Phone: 240-292-4241  
Email: elsafymm@nih.gov 
 
Clinical Project Manager: 
 
Effie Y.H. Nomicos, RN, MSN, CCRP 
NIH/NIAID/DMID 
5601 Fisher’s Lane, Room 8A35 
Bethesda, MD 20892 
Mail Stop 9825 
Phone: 240-627-3329 
BB: 240-507-0343 
Email:enomicos@mail.nih.gov 
 

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Statistical and Data Coordinating 
Center: 
 
The Emmes Corporation 
401 N. Washington St., Suite 700 
Rockville, MD 20850 
Phone: 301-251-1161 
Fax: 301-251-1355 
Email: parasitology@emmes.com 
 
Sonia Jain, PhD 
Co-Director, UCSD Biostatistics Research Center 
University of California, San Diego 
9500 Gilman Drive 
La Jolla, Ca 92093-0725 
Phone: 858-822-2388 
Email: sojain@ucsd.edu 
Regulatory Affairs Specialist: 
 
Blossom T. Smith, MS 
Office of Regulatory Affairs (HNM5G) 
National Institute of Allergy & Infectious Diseases 
National Institutes of Health 
BG 5601FL RM 7F45 
Mail Stop 9826 
5601 Fishers Lane 
Bethesda, MD 20852 
Tel: 240-627-3376 
E-mail:  blossom.smith@nih.gov 
 

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2 
BACKGROUND INFORMATION AND SCIENTIFIC 
RATIONALE 
2.1 
Background Information 
2.1.1 Summary of Previous Pre-clinical Studies 
This proposed clinical trial follows from key findings in studies supported by UO1 AI0778822 
entitled, “Novel Therapeutics for Class B Protozoa,” to design and develop highly effective 
antiparasitic agents for Class B Protozoa. The development of the first high-throughput whole 
cell screens for E. histolytica (Debnath, 2012) and G. lamblia (Gut et al., 2011) against FDA-
approved drugs and bioactive compounds (a “repurposing” screen), found that auranofin had a 
ten-fold lower IC50 (0.5M) compared with metronidazole (5.2 M) for E. histolytica and an 
equivalent IC50 for Giardia (4.0 M). Auranofin is an orally available gold-containing 
compound that has been in clinical use in the treatment of rheumatoid arthritis for 25 years.  The 
ability to reprofile an existing drug will significantly shorten the time and expense required to 
develop a drug against these important parasitic infections (Ashburn and Thor, 2004). 
 
In most organisms there are two largely independent systems to detoxify reactive oxygen species, 
one based on glutathione and the other based on thioredoxin, but E. histolytica and Giardia lack 
both glutathione reductase activity and glutathione synthetic enzymes (Fahey et al, 1984).  So the 
thioredoxin reductase is a major defense in both parasites in the prevention, intervention and 
repair of damage caused by oxidative stress (Arias et al., 2007). Auranofin inhibited recombinant 
EhTrxR with an IC50 of 400 nM (Debnath, 2012) and recombinant GiardiaTrxR with an IC50 of 
150 nM (Tejman-Yarden, 2013). In addition, inhibition of EhTrxRled to increased susceptibility 
of the trophozoites to oxidants as well as the accumulation of intracellular reactive oxide species.  
We also showed that auranofin caused the accumulation of oxidized thioredoxin in amebic 
trophozoites in vitro and in vivo. Therefore, auranofin is the first drug active against Entamoeba 
and Giardia with a clearly defined target. Based on these studies, auranofin was given Orphan 
Drug status by the FDA. 
 
Activity of auranofin against metronidazole-sensitive and resistant strains of Giardia. 
The drug susceptibility of a metronidazole sensitive, Assemblage A strains (WB (ATCC 50803) 
compared to their isogenic metronidazole resistant strains (WB-M1, WB-M2). The viability of 2 
X 103 trophozoites in 96 well plates with auranofin concentrations of 5 nM -100M and 
metronidazole concentrations of 0.1 M-2 mM was determined at 48 hours in a luminescence-
based ATP assay (Tejman-Yarden, 2013). Auranofin had equivalent efficacy with Mtz-sensitive 

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10 
and resistant strains, whereas the EC50 for Mtz-resistant strains was increased 8-40 fold (Figure 
1). 
Figure 1: Killing of metronidazole-sensitive and resistant strains by metronidazole (Mz) 
and auranofin (AF)  
 
 
The % viability of a sensitive parental strain (WB) and two resistant strains (WB-M1 and M2) 
to serial dilutions of metronidazole and auranofin are shown. 
Efficacy of auranofin in animal models of amebiasis and giardiasis. Auranofin was effective in 
an in vivo model of murine amebic colitis, in which trophozoites invade the mouse cecum 
following surgical inoculation (Debnath, 2012). Auranofin was delivered by gavage at a 
concentration of 1 mg/kg for 7 days.  The parasite burden of auranofin-treated mice by 
quantitative PCR significantly decreased compared to the control (Figure 2A) in contrast to 
metronidazole (Figure 2B), which required ten times the dose for a significant effect. 
 
 
The treatment of mice with (A) auranofin at 1 mg/kg daily dose or (B) 
metronidazole is presented as the percentage of trophozoites/ gm of tissue. 
 
The efficacy of auranofin was also tested in the suckling mouse models of giardiasis. Newborn 
C57BL/6 mice (5-7 days old) were gavaged with 107 G. lamblia trophozoites (Assemblage A 
 
Figure 2: Effect of auranofin on amebic colitis in mice.  

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11 
strain WB or Assemblage B strain 1279, infection established over two days, and then treated 
daily with 5 mg/kg auranofin for 5 days. The number of trophozoites remaining after drug 
treatment was determined by direct counts of the small intestinal contents. Auranofin treatment 
significantly decreased the trophozoite load (Figure 3).  
 
 
 
 
 
 
 
 
 
 
Newborn (5-7 days) or adult mice were infected with trophozoites of strains 106, 1279, or GS/M 
and auranofin started 2 days afterwards for 5 days. Values represent the log number of 
trophozoites present in the small intestine. 
 
Efficacy of auranofin against cysts. We also tested the efficacy of auranofin against Entamoeba 
cysts as E. histolytica cyst are resistant to metronidazole (Upcroft, 2001). Because E.histolytica 
cannot be induced to encyst in vitro, we used E. invadens, which do. Viability was tested with a 
fluorescent ATP assay (GoLive). E. invadens cysts were resistant to metronidazole, in contrast to 
auranofin (Figure 4). We will test the in vitro efficacy of auranofin against clinical E. histolytica 
cysts both in vitro and in treated patients in this study.  
 
Figure 3: Efficacy of auranofin in rodent models of giardiasis 

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12 
 
 
 
 
 
 
 
 
 
 
 
 
 
Viability was determined as the % survival compared to controls in ethanol alone.  
2.1.2 Summary of Relevant Clinical Studies 
The hypothesis is that auranofin/Ridaura will be an important drug for use against metronidazole 
resistant parasites, particularly Entamoeba histolytica, Giardia, and Trichomonas.  We have both 
in vitro and in vivo data supporting this therapeutic hypothesis. The FDA approved auranofin in 
1985 with limited PK/PD data because current sensitive techniques such as mass spectrometry 
were not available. To fill this gap, DMID performed a Phase I, single center, open label, multiple 
dose study to evaluate the PK of gold, administered as auranofin (Clinical Study Report 12-0101, 
finalized May 13, 2015). Fifteen healthy subjects were enrolled to receive the same treatment 
dose (6 mg daily for 7 days) as proposed for amebiasis in this study.  The pharmacokinetics of 
gold was evaluated by Inductively Coupled Plasma-Mass Spectroscopy (ICP-MS) in plasma (up 
to Day 126) and in feces (up to Day 42). Treatment-emergent adverse events (TEAEs) were 
monitored from Day 1 until Day 14 and serious adverse events (SAEs) until Day 126. All subjects 
(15) were included in the summary analyses for fecal gold, but two subjects were excluded (13 
final) from summary analyses of plasma gold concentration because of quantifiable Day 1 pre-
dose plasma gold concentrations higher than 5% of their Cmax.  
All subjects had quantifiable plasma gold levels throughout the 24 hour dosing period with a Tmax 
at 1.5 post dose. The plasma AUC(0-24) increased approximately six-fold from 0.971 + 0.51 to 
5.05 +1.07 g-h/mL and the Cmax three-fold from 0.102 + 0.04 to 0.312 +0.08 g/mL.  On Day 
7, 11 of 15 subjects had fecal gold concentrations of 8.80 + 7.83 mg/kg and undetectable levels 
at Day 14. The study concluded that auranofin at 6 mg per day dose for 7 days was generally safe 
Figure 4: Efficacy of auranofin and metronidazole against Entamoeba cysts 

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13 
and well tolerated with no deaths or SAEs. There were no clinically notable TEAEs or subject 
withdrawn from the study (see 2.3.1 for more details).  
2.1.3 Summary of Epidemiological Data 
Up to 10% of the world's population is infected with Entamoeba, resulting in morbidity second 
only to malaria and schistosomiasis among parasitic infections (Amoebiasis, 1997). Infection is 
initiated by the ingestion of environmentally resistant cysts in contaminated water or food, which 
release motile trophozoites in the intestine.  E. histolytica trophozoites can then invade either the 
mucosa to cause symptomatic colitis, or the bloodstream to cause distant abscesses of the liver, 
lungs, or brain (Haque et al., 2003). In endemic areas such as our clinical site in Bangladesh, 40% 
of children develop amebic colitis a year (Haque, 2006).  
Giardiasis is the most common gastrointestinal parasitic infection in the U.S. (Yoder, 2010). In 
developing countries, giardiasis is a major public health problem with infection rates in many 
regions as high as 15-20% in adults and close to 100% in children (Gilman et al., 1985), leading 
to dehydration and malnutrition. Because of its worldwide prevalence, Giardia was added to the 
WHO Neglected Disease Initiative (Savioli et al., 2006). 
2.2 
Rationale 
Entamoeba histolytica and Giardia lamblia are Class B protozoa, which cause major water- and 
foodborne outbreaks worldwide. Infection is caused by the ingestion of environmentally resistant 
cysts in water or contaminated food. Everyone in the U.S. is susceptible to these parasites, and at 
least six waterborne outbreaks of amebiasis have been documented in the U.S. and 20,000 cases 
of giardiasis reported a year.  Drug resistance is also an increasing concern with the majority of 
patients worldwide treated with imidazoles, particularly metronidazole, which causes cross-
resistance to the newest approved drugs, tinidazole and nitazoxanide. Metronidazole has frequent 
side effects, is carcinogenic and mutagenic in animals, and does not kill cysts. Therefore, shorter, 
more tolerable, and cyst killing therapy is needed.  
 
This proposed clinical trial will test the hypothesis that oral auranofin is effective for the 
treatment of amebiasis and giardiasis and follows from studies supported by UO1 AI0778822 
entitled, “Novel Therapeutics for Class B Protozoa.” The overall aim of the project was to 
design and develop highly effective antiparasitic agents for Class B Protozoa. As part of whole-
cell screening of amebic trophozoites against FDA-approved drugs, one drug, auranofin, was 
found to have an IC50 of 0.5M vs. the standard drug, metronidazole (IC50= 5.2 M). We 
found that auranofin targets the E. histolytica thioredoxin reductase (EhTrxR) in vitro and is 
efficacious against amebic colitis and liver abscess in rodent models. Studies with Giardia 
demonstrated that auranofin was very effective against both metronidazole-sensitive and 
resistant strains of Giardia in vitro and in rodent models. Auranofin is an orally available gold-

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14 
containing compound, FDA-approved in 1985 for rheumatoid arthritis, which has received 
Orphan Drug status for the treatment of amebiasis. During the planning stage of this trial, 
funded by R34 Grant AI098633-01, we have identified the International Centre for Diarrheal 
Diseases in Bangladesh (icddr,b) as the ideal clinical site under the direction of Dr. Rashidul 
Haque with Dr. William Petri as Co-PI. Potential patients are routinely screened for both E. 
histolytica and G. lamblia, so we can efficiently enroll patients infected with either parasite, 
supporting potential approval for two indications for auranofin in a single trial. This is an NIH 
Priority as the use of auranofin for amebiasis and giardiasis has been awarded both an R34 
Planning Grant as well as a Phase I Clinical Trial Grant. 
Because this trial will re-profile an FDA-approved drug that is still in use, we propose a Phase 
IIa, randomized, single-blinded, placebo-controlled superiority clinical trial comparing auranofin 
(standard dose of 6 mg daily) for 7 days for treatment of amebiasis or 5 days for giardiasis to 
placebo in adults. The two primary outcomes will be the proportion of patients with resolution of 
amebiasis by day 7 of therapy and/or resolution of giardiasis by day 5.  Any patients who remains 
asymptomatic but stool positive at 28 days will receive standard of care therapy with 
metronidazole. 
2.3 
Potential Risks and Benefits 
2.3.1 Potential Risks 
Auranofin is contraindicated in patients with a gold allergy, and not recommended during 
pregnancy or severe hepatic or renal insufficiency. The only reported drug interaction is a single 
patient with elevated phenytoin blood levels. The complications listed in the package insert for 
long term (> 1 yr) auranofin therapy include dermatologic: rash in 26%; gastrointestinal: loose 
stools (42%), abdominal pain (14%), nausea (10%); hematologic: anemia, leucopenia, 
thrombocytopenia in up to 3%; hepatic: elevated liver enzymes: 2%; mucous membranes: 
stomatitis (13%); renal: proteinuria (1%).  
In the DMID-sponsored Phase I Clinical Trial 12-0101 (Section 2.1.2), the safety of auranofin 
was assessed in 15 healthy volunteers based on adverse events collected from the first dose on 
Day 1 through Day 14 and through Day 126 for SAEs. Four subjects (26.7%) reported Treatment-
related TEAEs, one with headache, one with headache and nausea, one with abdominal 
discomfort and flatulence, and one with diarrhea. All were assessed as mild by the investigator. 
There were no clinically meaningful trends or changes in clinical laboratory results, vital signs, 
or physical examination data during the study.  
Auranofin is a Category C drug whose use is not recommended in pregnant women because of 
studies in pregnant rabbits and rats with decreased maternal and fetal weight and litter size with 

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15 
4-50X the human daily dose. The risk to pregnant women in our trial will be mitigated by careful 
screening, pregnancy testing and providing contraception (hormonal oral or injectable 
contraceptives for women in addition to providing condoms for their partners) for the length of 
the trial (5-7 days) and for three half-lives of the drug (total 4 months). Birth control method and 
compliance will be assessed during screening and throughout the subject’s participation. 
2.3.2 Known Potential Benefits 
Treatment worldwide for giardiasis relies primarily on imidazoles, particularly metronidazole, 
which was first introduced in 1959. Metronidazole has not been approved for use against Giardia 
in the U.S. and has been associated with treatment failures up to 20% (Upcroft 2001). It also 
causes frequent side effects, including a disulfiram-like effect (severe nausea and vomiting with 
alcohol ingestion), central and peripheral nervous system abnormalities (seizures, encephalopathy 
and peripheral neuropathy), and interactions with drugs metabolized by the CYP2C9 enzyme of 
the P450 system (Freeman et al., 1997). Metronidazole is also carcinogenic and teratogenic in 
animals (Bendesky et al., 2002). Nitazoxanide, a thiazolide drug, is the first drug with efficacy 
against Cryptosporidium as well as Giardia compared to placebo (Rossignol et al., 2001). 
Albendazole therapy of giardiasis has been evaluated in several clinical trials (reviewed in 
Solaymani-Mohammadi et al. 2010.) Quinacrine and Furazolidone also have activity, but are no 
longer available in the U.S. (Gardner and Hill, 2001). Metronidazole resistance is now a major 
concern as clinical failures have been linked to metronidazole-resistant Giardia (Upcroft et al., 
2001; Lemee et al., 2000), and highly resistant strains are readily produced in the laboratory 
(Dunn et al., 2010). Unfortunately, metronidazole-resistant Giardia are cross- resistant to 
nitazoxanide (Wright et al., 2003) and all metronidazole derivatives. 
In addition, auranofin may prove to be a future broad spectrum antiparasitic drug as in vitro and/or 
in vivo efficacy has already been demonstrated against amebiasis (Debnath, 2012), trichomoniasis 
(Dr. Kirk Land, University of the Pacific, personal communication), Cryptosporidium (Dr. 
Momar Ndao, personal communication), Toxoplasma gondii (Andrade, 2014), T. brucei 
(Lobanov, 2006), Leishmania infantum (Ilari, 2011), filaria (Dr. James McKerrow, UCSF, 
personal communication), and schistosomiasis (Kuntz, 2007; Angelucci, 2009). 

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3 
OBJECTIVES 
3.1 Study Objectives 
Auranofin is a “repurposed” drug with established pharmacology and is approved for human use 
by the FDA.  We will assess clinical efficacy for two new indications, amebiasis and giardiasis, 
with a randomized Phase IIa, placebo-controlled, single-blinded, superiority trial comparing 
auranofin for treatment of amebiasis and giardiasis to placebo in adults. Resolution of diarrhea 
and clearance of parasites will be the endpoints, and all subjects who remain positive at Day 28 
for giardiasis or amebiasis will receive standard of care treatment with metronidazole. Ultimately, 
the goal will be to expand the trial to include symptomatic children after proving efficacy and 
safety in adults. 
 
All subjects will have the same screening protocol, but the trial will evaluate subjects with 
amebiasis or giardiasis with separate primary and secondary endpoints.  
 
Asymptomatic subjects enrolled under prior versions of the protocol will be analyzed as per 
the corresponding objectives and study SAP.  
 
Primary E. histolytica: 
To compare the proportion of subjects with stools positive by rapid EIA and positive antigen 
detection EIA for E. histolytica at enrollment with resolution of diarrhea (less than 3 loose stools/ 
24 hours) by Day 7.  
Secondary E. histolytica: 
 
• To compare the proportion of subjects with positive rapid EIA and positive antigen 
detection EIA for E. histolytica and trophozoites on smear (wet mount or concentrated 
trichrome) at enrollment with parasitological response (no detection of trophozoites on 
microscopic exam by Day 7.  
• To compare the proportion of subjects with positive rapid EIA and positive antigen 
detection EIA for E. histolytica and trophozoites on smear at enrollment with 
parasitological response (no detection of trophozoites on microscopic exam) by Day 3 
and 5. 
• To compare the rate of decrease in trophozoite/cyst load by qPCR in stools by Days 3, 5, 
and 7. 
• To compare the proportion of subjects with negative stool antigen test by Days 3, 5, 7, 
and 14. 

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• To compare the proportion of subjects with sustained cure (no detection of trophozoites 
by microscopic exam) at 14 and 28 days. 
• To compare the proportion of subjects with relapse (same strain) or re-infection (new 
strain) with positive stools at 14 and/or 28 days by genotyping the initial vs. subsequent 
strain. 
• To compare the time to resolution of diarrhea (less than 3 loose stools/24 hours) 
 
 
Primary Giardia: 
 
To compare the proportion of subjects with stool positive rapid EIA and positive antigen detection 
EIA for Giardia at enrollment with resolution of diarrhea (less than 3 loose stools/24 hours) by 
Day 5. 
Secondary Giardia: 
• To compare the proportion of subjects with parasitological response (no 
detection of trophozoites on microscopic exam) by Day 3 and 5. 
• To compare the rate of decrease in trophozoite/cyst load by qPCR in stools by 
Days 3 and 5. 
• To compare the proportion of subjects with negative stool antigens by days 3 
and 5. 
• To compare the proportion of subjects with sustained cure (no detection of 
trophozoites by microscopic exam) at 14 and 28 days. 
• To compare the proportion of subjects with relapse (same strain) or re-
infection (new strain) with positive stools at 14 and/or 28 days by genotyping 
the initial vs. subsequent strain. 
• To compare the time to resolution of diarrhea (less than 3 loose stools/24 
hours) 
 
3.2 
Study Outcome Measures 
3.2.1 Primary Outcome Measures 
1. Proportion of subjects with positive rapid EIA and positive antigen detection EIA for E. 
histolytica and resolution of diarrhea (less than 3 loose stools/ 24 hours) by Day 7.  
2. Proportion of subjects with positive rapid EIA and positive antigen detection EIA for 
Giardia and resolution of diarrhea (less than 3 loose stools/24 hours) by Day 5.  

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3.2.2 Secondary Outcome Measures 
1. E. histolytica: 
a. Proportion of subjects with positive rapid EIA and positive antigen 
detection EIA for E. histolytica and trophozoites on smear at enrollment 
with parasitological response (no detection of trophozoites on microscopic 
exam) by Day 7. 
b. Proportion of subjects with positive rapid EIA and positive antigen 
detection EIA for E. histolytica and trophozoites on smear at enrollment 
with parasitological response (no detection of trophozoites on microscopic 
exam) by Day 3 and 5. 
c. Rate of decrease of trophozoite/cyst load by qPCR in stools by Days 3, 5, 
and 7 
d. Proportion of subjects with negative stool antigen test by Days 3, 5, 7, and 
14 
e. Proportion of subjects with sustained cure (no detection of trophozoites by 
microscopic exam) at 14 and 28 days  
f. 
Proportion of subjects with relapse (same strain) or re-infection (new 
strain) with positive stools at 14 and 28 days by genotyping the initial vs. 
subsequent strain 
g. 
Time to resolution of diarrhea (less than 3 loose stools/24 hours) 
 
2.  Giardia: 
a. Proportion of subjects with positive rapid EIA and positive antigen 
detection EIA for Giardia and trophozoites on smear at enrollment with 
parasitological response (no detection of trophozoites on microscopic 
exam) by Day 3 and 5  
b. Rate of decrease of trophozoite/cyst load by qPCR in stools by Days 3 and 
5 
c. Proportion of subjects with negative stool antigens by days 3 and 5.  
d. Proportion of subjects with sustained cure (no detection of trophozoites by 
microscopic exam) at 14 and 28 days  
e. Proportion of subjects with relapse (same strain) or re-infection (new 
strain) with positive stools at 14 and 28 days by genotyping the initial vs. 
subsequent strain 
f. Time to resolution of diarrhea (less than 3 loose stools/24 hours) 

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4 
STUDY DESIGN 
This 30-day, Phase IIa, two-arm, randomized, placebo-controlled, single-blinded, superiority 
treatment study in 136 males and non-pregnant females ≥ 18 to 65 years of age will compare 
placebo to once daily doses of auranofin (Ridaura) for adults with amebiasis (68 subjects)  or 
giardiasis (68 subjects).Potential Bangladeshi adult subjects will be identified by a health 
professional from otherwise stable patients presenting to the icddr,b or Rajshahi Medical College 
Hospitals with diarrhea (thought to be due to amebiasis or giardiasis). Rajshahi Medical College 
Hospital is being added as a new enrollment site because of their higher prevalence of amebic 
infections (Alam et al., 2014). Eligible subjects will be randomly assigned to a treatment group 
with auranofin (6 mg orally once daily for 5 days for giardiasis or 7 days for amebiasis) compared 
to a placebo group receiving similar but not identical placebo capsules. Projected duration of 
subject participation will be approximately 30 days of face to face visits, including the pre-
enrollment screening period of up to 4 days. It is anticipated that it will take approximately 3.5 
years to finish the study.   
Primary and secondary outcomes to be measured during the course of the study are outlined in 
Section 3.2. 
For additional details on study procedures and evaluations by study schedule and study visits see 
Section 7, 8, and Appendix A. 
4.1 
Sub Studies 
No sub studies are planned. 

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5 
STUDY ENROLLMENT AND WITHDRAWAL 
One hundred thirty six males and non-pregnant females, aged 18-65 years, with amebiasis or 
giardiasis who are otherwise healthy and meet all eligibility criteria, will be chosen from adult 
patients presenting to the iccdr,b  or Rajshahi Medical College Hospitals with mild diarrhea. 
 
Subject Inclusion and Exclusion Criteria must be assessed by a study clinician licensed to make 
medical diagnoses and listed on the Form FDA 1572 as the site principal investigator or sub-
investigator. 
 
No exemptions are granted on Subject Inclusion/Exclusion Criteria in DMID-sponsored studies.  
Questions about eligibility should be directed toward the DMID Medical Officer. 
5.1 
Subject Inclusion Criteria 
Subjects eligible to participate in this study must meet all of the following inclusion criteria:  
1. Provide written informed consent prior to initiation of any study procedures. 
 
2. Able to understand and comply with planned study procedures and be available for all 
study visits. 
 
3. Male or non-pregnant, non-lactating females 18-65 years of age, inclusive. Females of 
reproductive potential currently using effective contraceptive methods are eligible. 
 
4. Amebiasis or giardiasis identified by rapid EIA and positive antigen detection EIA of 
stool1.1 
 
1If a subject is infected with both E. histolytica and Giardia, they will be enrolled in the 
E. histolytica study arm. Once the Entamoeba study arm is fully enrolled, any subsequent 
dual infected subjects will be enrolled in the Giardia arm.  If a subject is infected with 
both Giardia and Cryptosporidium, they will not be enrolled.  
 
5. Has diarrhea (defined as three or more loose stools) in the past 24 hrs, but is assessed to 
be clinically stable and in otherwise good health2. 
2As determined by medical history and targeted physical examination, if indicated based 
on medical history, to evaluate acute or currently ongoing chronic medical diagnoses or 
conditions that would affect the assessment of eligibility and safety of subjects. Existing 

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medical diagnoses or conditions (except those in the Subject Exclusion Criteria) must be 
deemed as stable chronic medical conditions. A stable chronic medical condition is 
defined as no change in prescription medication, dose, or frequency of medication in the 
last 3 months (90 days) and health outcomes of the specific disease are considered to be 
within acceptable limits in the last 6 months (180 days).  Any change due to change of 
health care provider, insurance company, or that is done for financial reasons, as long as 
in the same class of medication, will not be considered a violation of this inclusion 
criterion.  Any change in prescription medication due to improvement of a disease 
outcome, as determined by the site principal investigator or appropriate sub-investigator, 
will not be considered a violation of this inclusion criterion. Subjects may be on chronic 
or as needed (prn) medications if, in the opinion of the site principal investigator or 
appropriate sub-investigator, they pose no additional risk to subject safety. Topical, nasal, 
and inhaled medications, vitamins, and contraceptives are permitted. 
 
6. Vital signs (oral temperature, pulse, and blood pressure) are all within normal protocol-
defined ranges (abnormal criteria defined in Section 9.2.3 Additional Adverse Event 
Severity Grading). 
 
7. Laboratory tests (blood urea nitrogen, creatinine, AST, ALT, white blood cells, platelets, 
hemoglobin) are all within protocol-defined ranges. 
 
Subjects will be eligible for enrollment with the following laboratory values:  
• Blood urea nitrogen less than or equal to 30 mg/dL 
• Creatinine less than or equal to 133umol/L 
• AST or ALT less than or equal to 70.0 U/L 
• White cell count between 3.5 and 13.0 inclusive(109/L) 
• Platelets between 131 and 550 (109/L) inclusive 
• Hemoglobin between 11.0 and 18.0 gm/dL inclusive 
 
8. Urinalysis with no greater than trace protein. If a high protein is confirmed to be due to 
menstruation, it should be repeated. 
 
9. Women of reproductive potential4 must have a negative urine pregnancy test within 72 
hours of starting study medications.  
 
4Female subjects who are surgically sterile via tubal sterilization, bilateral oophorectomy 
or hysterectomy, who have been postmenopausal for greater than 1 year are not considered 
to be of reproductive potential. 

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10. Female subjects participating in sexual activity that could lead to pregnancy must be using 
and continue to use highly effective5 contraception for a total of 4 months after enrollment.   
 
5Highly effective methods of contraception are defined as having low failure rates (i.e. 
less than 1% per year) when used consistently and correctly and may include, but are not 
limited to, abstinence from intercourse, monogamous relationship with a vasectomized 
partner, male condoms with spermicide, diaphragm with spermicide, intrauterine devices, 
and licensed hormonal methods.  Females on effective forms of birth control will continue 
while on the study and for the follow-up period of 4 months total. The method and 
compliance of birth control used will be confirmed and documented at all study visits. 
5.2 
Subject Exclusion Criteria 
Subjects meeting any of the following exclusion criteria at baseline will be excluded from study 
participation within 1 week of the initial visit: 
1. Known intolerance of auranofin or gold compounds. 
 
2. Pregnant or breastfeeding women or women of reproductive potential not using effective 
contraception or who plan to become pregnant or breastfeed at any given time during the 
study or within 3 months of study completion. 
 
3. Use of metronidazole within the past 7 days. 
 
4. Has any condition that would, in the opinion of the site investigator, place the subject at an 
unacceptable risk of injury or render the subject unable to meet the requirements of the 
protocol. 
 
5. Concurrent participation in other investigational protocols or receipt of an investigational 
product within the previous 30 days. 
 
6. History of alcohol or drug abuse within the last five years. 
 

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5.3 
Treatment Assignment Procedures 
5.3.1 Enrollment and Randomization Procedures 
Per International Conference on Harmonisation (ICH) guideline E6: Good Clinical Practice 
(GCP), screening records will be kept at the icddr,b or Rajshahi Medical College hospital  site to 
document the reason why an individual was screened, but failed trial entry criteria.  The reasons 
why individuals failed screening will be recorded in the Statistical and Data Coordinating 
Center’s (SDCC) Advantage EDCSM (Electronic Data Capture System). No subject may be 
screened more than twice due to a screening failure result.  
 
Randomization will be stratified by site to prevent confounding that could arise from imbalance 
in treatment allocations between sites.  Because the relative pace of enrollment between the two 
sites is unknown, the number of participants to be enrolled at each site is not fixed in advance.  
With this approach, the faster enrolling site may enroll more subjects, allowing enrollment to 
complete as quickly as possible.  Each site may enroll up to 68 subjects into each strata, so it is 
possible that one strata could be filled entirely with subjects from a single site.  With the flexible 
approach, treatment assignments may not be perfectly balanced (overall or within each site), but 
they will be approximately balanced.  With sample sizes of 68 in each strata, the impact of a small 
imbalance on power is negligible.   
Once eligibility and consent are confirmed, subjects will be stratified by site and infection (a 2-
level variable of either amebiasis vs. giardiasis), and then within each stratum, randomized 1:1 to 
receive auranofin or placebo on Day 1. Both disease groups will enroll concurrently.  Enrollment 
will continue in the other arm after one arm reaches full enrollment. All subjects will receive 
doses of the assigned medication or placebo delivered orally on Days 1, 2, 3, 4, 5, 6, and 7 for 
amebiasis and Days 1, 2, 3, 4, and 5 for giardiasis. 
The randomization code will be prepared by statisticians at the SDCC and included in the 
enrollment module for the trial. SDCC staff will generate two randomization lists and send 
corresponding documentation to the study site. The randomization lists will include the 
randomization codes that will be used to serially assign study treatment to subjects as they are 
assigned to the study (i.e., randomize subjects to study arm by study identification number).  This 
table will not contain unblinded information; study staff will not be able to see the study arm 
assignment for each study identification number.  
Eligible study subjects will be randomized under single-blind conditions by study staff members 
after consent. Since the placebo is similar but not identical to the study medication, only the lab 

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personnel are truly blind, however the participant will not be informed whether they receive study 
product or placebo. The randomization number will be recorded on the study CRFs. 
A designated individual at icddr,b and Rajshahi Medical College hospital  will be provided with 
the treatment key, which links the treatment code to the actual treatment assignment, which will 
be kept in a secure place.  
5.3.2 Masking Procedures 
This is a single-blind study because the placebo is similar, but not identical to the study drug. The 
laboratory personnel performing evaluations will be blinded to treatment assignment. 
Subjects, investigators, and study personnel performing any study-related assessments following 
study product administration will not be informed of group allocation, but could detect differences 
in study and placebo capsules. They will be instructed to perform their clinical or AE assessments 
as though blinded to prevent potential bias.  
The randomization scheme will be generated by the SDCC and provided to unblinded study 
personnel (i.e., study pharmacist).  
The Data and Safety Monitoring Board (DSMB) may receive data in aggregate and presented by 
treatment group.  The DSMB may also be provided with expected and observed rates of the 
expected AEs in an unblinded fashion.  The DSMB may be unblinded to individual study 
treatment assignments, as needed, to adequately assess safety issues. Refer to the MOP for 
unblinding procedures. 
5.3.3 Reasons for Withdrawal 
Subjects may voluntarily withdraw their consent for study participation at any time and for any 
reason, without penalty. Subjects who have received study product, regardless of the number of 
doses received, or who developed an AE or SAE will be encouraged to remain in the study to be 
followed for safety purposes. 
A subject may withdraw or be withdrawn from this study for any of the following reasons: 
• Medical disease or condition, or any new clinical findings for which continued 
participation, in the opinion of the site principal investigator or appropriate sub-
investigator, would compromise the safety of the subject, or would interfere with the 
subject's successful completion of the study, or would interfere with the evaluation of 
responses. 

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• Subject no longer meets eligibility criteria. 
• As deemed necessary by the site principal investigator or appropriate sub-investigator for 
noncompliance or other reasons. 
• Subject withdrawal of consent. 
• Subject lost to follow-up. 
• Termination of the study. 
• New information becomes available that makes further participation unsafe. 
A subject will be discontinued from further receipt of auranofin or placebo if: 
• The subject misses more than one dose of drug or placebo.  
• Any clinical adverse event, laboratory abnormality, intercurrent illness, other medical 
condition or situation occurs such that continued participation in the study would not be 
in the best interest of the subject. 
5.3.4 
Handling of Withdrawals 
The primary reason for withdrawal from the study will be recorded on the Study Status data 
collection form.  Subjects will be encouraged to complete the Early Termination Visit.  The Early 
Termination Visit procedures are listed in Section 7.4.  Although subjects are free to withdraw at 
any time or may be withdrawn by the site principal investigator or appropriate sub-investigator at 
any time, subjects who receive at least one dose of study product will be encouraged to remain in 
the study for follow-up safety assessments and collection of stool specimens.  Every attempt will 
be made to follow all adverse events, including systemic reactions, serious adverse events, and 
new-onset chronic medical conditions ongoing at the time of early withdrawal to resolution. 
In the case of subjects who fail to appear for a follow-up safety assessment, extensive effort (i.e., 
three documented contact attempts via phone calls and/or in person visits to the household of the 
participants) will be made to locate or recall them, or at least to determine their health status. 
These efforts will be documented in the subject’s records. 
Subjects who sign the informed consent form and are randomized but do not receive study product 
may be replaced with new subjects who will receive the same treatment assignment. .  Subjects 
who sign the informed consent form, are randomized and receive study product, and subsequently 

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withdraw, or are withdrawn or terminated from this study, or are lost to follow-up will not be 
replaced.   
5.3.5 Termination of Study 
Although the study sponsor has every intention of completing the study, it reserves the right to 
terminate the study at any time for clinical or administrative reasons.  Reasons for termination 
include, but are not limited to, study closure due to DSMB review and recommendation and at 
the discretion of DMID. 
 

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6 
STUDY INTERVENTION/INVESTIGATIONAL PRODUCT 
6.1 
Study Product Description 
Auranofin (2,3,4,6-tetra-O-acetyl-1-thio-D-glucopyranosato-S) (triethyl-phosphine gold), is an 
orally available gold-containing compound with established pharmacology and is approved for 
human use by the FDA. It is available in an oral form as capsules called Ridaura, which contains 
3 mg of auranofin. Auranofin 3 mg capsules are manufactured by Prometheus Laboratories, San 
Diego, CA. 
6.1.1 Acquisition 
Auranofin 3mg capsules will be purchased from a commercial supplier by Fisher BioServices. 
The placebo will be manufactured by commercial supplier through contract with Fisher 
BioServices. 
Upon request by DMID, Auranofin and placebo will be shipped to the following address: 
DMID-Clinical Material Services (CMS)  
Fisher BioServices 
20439 Seneca Meadows Parkway 
Germantown, MD 20876 
Phone: 240-477-1350 
Fax: 240-477-1360 
Email: DMID.CMS@thermofisher.com  
Auranofin and placebo will be stored at the DMID CMS- Fisher BioServices and will be provided 
to the clinical research site upon request and with prior approval from DMID. 
6.1.2 Formulation, Packaging, and Labeling 
6.1.2.1 
 
Auranofin Capsules 
Each capsule of auranofin has an opaque brown cap and opaque tan body, contains 3 mg of white 
crystalline powder, and is imprinted with the product name RIDAURA.2 Inactive ingredients 
consist of benzyl alcohol, cellulose, cetylpyridinium chloride, D&C Red No. 33, FD&C Blue No. 
1, FD&C Red No. 40, FD&C Yellow No. 6, gelatin, lactose, magnesium stearate, povidone, 
sodium lauryl sulfate, sodium starch glycolate, starch, titanium dioxide and trace amounts of other 
inactive ingredients. Auranofin 3 mg capsules are packaged in bottles of 60 count. 
 

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6.1.2.2 
 
Placebo Capsules 
The placebo capsules will be similar to auranofin capsules with opaque brown caps and an opaque 
tan body, but will not be imprinted with the product name RIDAURA. The placebo capsules will 
be manufactured by a contract supplier through Fisher BioServices. Measures will be taken to 
mitigate potential bias by the single-blinded nature of the trial. The specific measures will be 
detailed in the MOP.  
 
6.1.2.3 
Packaging and Labeling 
The daily dose of placebo and Auranofin capsules will be identical in a tight, light-resistant 
container. The study products will be labeled to include FDA cautionary information: “Caution- 
New drug -Limited by Federal (or United States) Law to Investigational Use Only.”    
 
6.1.2.4 
Storage and Stability 
The study drugs will be sent first to icddr,b and then transported by icddr,b staff under 
continuously monitored, temperature controlled conditions to Rajshahi Medical College hospital 
for storage and dispensing as detailed in the MOP. Auranofin powder darkens slightly when 
exposed to strong light and also to some extent when stored at a temperature of 60°C or warmer; 
the darkening indicates a small degree of chemical degradation, but the effect on biologic activity 
is not known. The Auranofin capsules and the placebo will be stored between 15° and 30°C (59° 
and 86°F).  
6.2 
Dosage, Preparation and Administration of Study 
Intervention/Investigational Product 
Auranofin 6mg (administered orally as two-3mg capsules) or similar placebo (administered orally 
as two capsules) once daily for 5 days for giardiasis or 7 days for amebiasis. All shipped study 
products will be initially stored at icddr,b hospital. Study products will subsequently be 
transported to Rajshahi Hospital for storage and dispensing. 
Enrollment may occur any day of the week. The complete course of study capsules (7 days for 
amebiasis and 5 days for giardiasis) will be dispensed in individual daily containers upon 
randomization by the icddr,b or Rajshahi pharmacy and dispensed as follows: 
For subjects enrolled at the icddr,b and Rajshahi Medical College Hospitals, the first doses will 
be dispensed until the patient is discharged, with all doses thereafter managed through home visits 
or at  icddr,b or Rajshahi Medical College Hospitals or local site clinic. Refer to the protocol-
specific MOP for additional information regarding dispensing procedures. 

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For all subjects: Friday is a holiday in Bangladesh so two days’ supply will be given on Thursday 
and the Friday dose will be self-administered as instructed and documented on the next visit.  
6.3 
Modification of Study Intervention/Investigational Product for a 
Participant 
Because auranofin is an FDA-approved drug, which has undergone extensive short term toxicity 
studies in a Phase I trial, there will be no dose modification of the drug, and subjects will be 
withdrawn if they miss more than one dose. If at baseline, a subject has any exclusionary 
abnormality in the screening blood and urine tests, they will be notified and not enrolled. The 
next safety lab testing time will be at Day 7 after completion of the study agent. Any laboratory 
abnormalities at that point will be evaluated as an AE, and may be reported to the DSMB as 
requested, and followed accordingly.  
6.4 
Accountability Procedures for the Study Intervention/Investigational 
Product(s) 
After receipt of the study product, the site Principal investigator (PI) is responsible for distribution 
and disposition of these study products, and has ultimate responsibility for drug accountability.  
The site PI may delegate this responsibility to the site pharmacist for each site.  Study product 
will be distributed to the health care workers to be dispensed during the subject’s hospital stay 
and/or during daily subject visits at home or the Rajshahi Hospital or the local Rajshahi clinic. 
Study protocol records must be maintained and document logs of receipt, accountability, and 
storage temperature conditions at the icddr,b office or Rajshahi clinic.  These study product 
accountability and dispensing logs must be maintained in the study file. Upon completion of the 
study and after the final monitoring visit, unused study product will be retained until monitored 
and released for disposition as per the Sponsor.  
6.5 
Assessment of Subject Compliance with Study 
Intervention/Investigational Product(s) 
Subject compliance will be assessed through direct observation and pill counts. 
6.6 
Concomitant Medications/Treatments 
Whenever a concomitant medication or study agent is initiated or the dose changed, investigators 
must review the concomitant medications’ and study agents’ most recent package inserts or 
investigator's brochure to obtain the most current information on drug interactions, 

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contraindications, and precautions. Administration of any medications, therapies, or vaccines will 
be recorded on the appropriate data collection form. Concomitant medications will include all 
current medications and medications taken within the previous 30 days through the end of the 
follow-up period and will be reported in the electronic case report form (eCRF). Prescription and 
over-the-counter drugs will be included as well as herbals, vitamins, and supplements.  
Use of new medication should prompt evaluation for the presence of a new diagnosis or chronic 
medical disease or condition. 
There are no drugs that are prohibited because of their interactions with Ridaura, but current 
systemic metronidazole are an exclusion criterion. There are no medications whose dose should 
be adjusted because of this short course of Ridaura treatment. Any patients with clinically 
significant underlying renal, hepatic, or immunologic abnormalities will be excluded before 
enrollment. 

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7 
STUDY SCHEDULE 
Complete study schedule details listed by study visit are described below. Any visit may occur 
at icddr,b or  Rajshahi Medical College Hospitals or local site clinic,  or home (except those 
requiring blood drawing, Visit 00B and Day 7). Refer also to Section 8 and Appendix A: 
Schedule of Events. 
7.1 
Screening Visits 
7.1.1 Visit 00A, Day -4 to -1, Screening 1, icddr,b or Rajshahi Medical 
College Hospitals 
Potential adult subjects from Bangladesh (between the ages of 18-65) will be chosen from adult 
patients presenting to the icddr,b or  Rajshahi Medical College Hospitals with mild diarrhea. 
These potential subjects will be visited by a study team member or health professional from 
icddr,b or   Rajshahi Medical College Hospitals. Screening may take place as many as 4 days 
prior to enrollment. A 5-minute screener will be administered at the first screening visit and will 
be used to identify eligible persons who will be referred to a study team member to discuss 
inclusion in the study. Written informed consent will be obtained from potentially eligible 
subjects after the 5-minute screener and prior to conducting any further screening or study 
procedures. A single ICF will be used to document the volunteer’s consent to further screening 
and participation in the study.  
For subjects who are potentially eligible and who have provided informed consent, the following 
procedures will be conducted at the first screening visit:  
• Full eligibility criteria will be reviewed with subjects.   
• Demographic data, including date of birth, sex, self-reported ethnicity, and residence 
location will be collected. 
• A stool sample will be collected for detection of Giardia or E. histolytica. 
7.1.2 Visit 00B, Day -4 to -1, Screening 2, icddr, b or  Rajshahi Medical 
College Hospitals   
Only eligible subjects who have provided informed consent and stool specimens that are positive 
for Giardia or E. histolytica will have this second screening visit for blood and urine testing. This 
visit may also occur on the same day at Visit 00A and must occur at the hospital or clinic.  

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• Medical history will be obtained by interview of subjects to assure eligibility.  
• All concomitant medications taken within 30 days prior to signing the informed consent 
form will be recorded on the appropriate data collection form and eCRF. Subjects will be 
questioned specifically about any metronidazole in the past 7 days.  
• A targeted physical examination will be performed, including vital signs (oral 
temperature, pulse (taken at the wrist or neck), blood pressure) observation for rashes or 
glossitis, and an abdominal exam.  
• A urine pregnancy test will be performed on all female subjects of childbearing potential 
and must be negative. 
• Approximately 17 mL of venous blood will be collected for CBC, ALT, AST, BUN, and 
creatinine tests. These values must be confirmed to meet the eligibility criteria as outlined 
in the Subject Inclusion Criteria (see Section 5.1) prior to randomization. 
• A urine sample will be collected to test for the presence of protein. The result must be 
confirmed to meet the eligibility criteria as outlined in the Subject Inclusion Criteria (see 
Section 5.1) prior to randomization. 
7.2 
Visit 01, Day 1, Enrollment, Dose 1, icddr,b or Rajshahi Medical 
College Hospital or Clinic 
• Enrollment and randomization of subjects 
• Eligibility criteria, including results of all clinical screening laboratory evaluations, will 
be reviewed with subjects to assure continued eligibility.  
• Targeted physical exam performed, if indicated 
• 2 capsules of study product (6mg total) or placebo will be orally taken by the subject.  
• Assessment of any new symptoms since Visit 00B before first dose is given. 
• Medical history, including any concomitant medications, reviewed.  
• Stool sample will be collected for ova and parasite testing. The window for stool 
specimens will be +1 day and the date of collection noted.   

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7.3 
Follow-up Visits 
This section summarizes the follow-up procedures conducted for each subject. Complete details 
for each Study Day are provided in Appendix A. Visit windows are specified around the target 
for each visit. Follow-up visits are scheduled in reference to dosing dates as indicated for each 
visit window. Subjects will be enrolled at the icddr,b or  Rajshahi Medical College Hospitals and 
have follow-up visits in the Hospital until their diarrhea has resolved. Subsequent follow-up visits 
may be held at the icddr,b, Rajshahi Hospital, or clinic or their home except for Visit 07 which 
must be at icddr,b or  Rajshahi Hospital or clinic because blood drawing is required. When a visit 
falls on a holiday Friday, two days’ supply of study product will be given on Thursday and the 
Friday dose will be self-administered as instructed and documented upon the next visit. Staff will 
notify the subject on Friday to take the dose and document it was taken. The window for stool 
specimens will be +1 day and the date of collection noted. For visits that fall on a Friday, the 
study team member will confirm on the next scheduled visit the diarrhea status as noted on the 
Visit Documentation for the missed visit.  This will be submitted in the Data System for study 
end-point analysis. 
7.3.1 Visit 02, Day 2 
• Targeted physical exam performed, if indicated.  
• 2 capsules of study product (6mg total) or placebo will be orally taken by the subject.  
• Assessment of any side effects or adverse events.  
• Any subject with worsening diarrhea as assessed by the study physician, may have their 
dose discontinued and receive standard of care metronidazole.   
• Medical history, including any concomitant medications, reviewed.  
7.3.2 Visit 03, Day 3 
• Targeted physical exam performed, if indicated.  
• 2 capsules of study product (6mg total) or placebo will be orally taken by the subject.  
• Assessment of any side effects or adverse events.  
• Any subject with worsening diarrhea as assessed by the study physician, may have their 
dose discontinued and receive standard of care metronidazole.   

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• Medical history, including any concomitant medications, reviewed.  
• Stool sample will be collected for ova and parasite testing. The window for stool 
specimens will be +1 day and the date of collection noted.   
7.3.3 Visit 04, Day 4 
• Targeted physical exam performed, if indicated.  
• 2 capsules of study product (6mg total) will be orally taken by the subject.  
• Assessment of any side effects or adverse events.  
• Any subject with worsening diarrhea as assessed by the study physician, may have their 
dose discontinued and receive standard of care metronidazole.   
• Medical history, including any concomitant medications, reviewed.  
7.3.4 Visit 05, Day 5 
• Targeted physical exam performed, if indicated.  
• 2 capsules of study product (6mg total) or placebo will be orally taken by the subject.  
• Assessment of any side effects or adverse events. 
• Any subject with worsening diarrhea as assessed by the study physician, may have their 
dose discontinued and receive standard of care metronidazole.    
• Medical history, including any concomitant medications, reviewed.  
• Stool sample will be collected for ova and parasite testing. The window for stool 
specimens will be +1 day and the date of collection noted.   
7.3.5 Visit 06, Day 6 (Amebiasis arm only) 
• Targeted physical exam performed, if indicated  
• 2 capsules of study product (6mg total) or placebo will be orally taken by the subject.  
• Assessment of any side effects or adverse events.  

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• Any subject with worsening diarrhea as assessed by the study physician, may have their 
dose discontinued and receive standard of care metronidazole.   
• Medical history, including any concomitant medications, reviewed.  
7.3.6 Visit 07, Day 7 (Window + 1 day) icddr,b or Rajshahi Medical 
College Hospitals 
• Targeted physical exam performed, if indicated.  
• 2 capsules of study product (6mg total) or placebo will be orally taken by the subject 
(amebiasis arm only).  
• Assessment of any side effects or adverse events.  
• Any subject with worsening diarrhea as assessed by the study physician, may have their 
dose discontinued and receive standard of care metronidazole.   
• Medical history, including any concomitant medications, reviewed.  
• Stool sample will be collected for ova and parasite testing (amebiasis patients only) The 
window for stool specimens will be +1 day and the date of collection noted.   
• Approximately 17 mL of venous blood will be collected for CBC, ALT, AST, BUN, and 
creatinine tests. The window for blood specimens will be +1 day and the date of collection 
noted.   
• A urine sample will be collected to test for the presence of protein.  
7.3.7 Visit 08, Day 14 (Window: +/- 3 days) 
• Targeted physical exam performed, if indicated.  
• Assessment of any side effects or adverse events. 
• Any subject with worsening diarrhea as assessed by the study physician, may have their 
dose discontinued and receive standard of care metronidazole.   
• Medical history, including any concomitant medications, reviewed.  

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• Stool sample will be collected for ova and parasite testing. The window for stool 
specimens will be +/- 3 days from the visit date and the date of collection noted.  
7.3.8  Visit 09, Day 28 (Window: +/- 3 days) 
The ninth study visit will occur on Day 28 when subjects will be assessed for medical history, 
targeted physical exam, concomitant medications review, and a final stool sample collected. 
Subjects will be instructed to contact the study staff, per the informed consent form, if any study 
product related adverse events develop in the following three months. 
7.3.9 Visit 10, Day 30 (Window: +/- 3 days)  
Visit 10 is a visit for any study patient who remains stool positive for Giardia or E. histolytica in 
the Day 28 stool specimen to receive the standard of care treatment for giardiasis or amebiasis. 
Subjects will be instructed to contact the study staff, per the informed consent form, if any adverse 
events develop in the following three months. 
7.4 
 Early Termination Visit 
 
Subjects may withdraw voluntarily from participation in the study at any time.  Subjects may also 
withdraw voluntarily from receiving the study intervention for any reason. If early termination 
occurs and if the subject is willing, a final assessment for symptoms will be performed and a stool 
specimen obtained.  
 
If voluntary withdrawal occurs, the subject will be asked to continue scheduled evaluations, 
complete an end-of-study evaluation, and be given appropriate care under medical supervision 
until the symptoms of any adverse event resolve or the subject’s condition becomes stable. 
7.5 
Unscheduled Visit 
The icddr,b and   Rajshahi Medical College Hospital Study Teams have local clinic facilities 
that are open six days a week for unscheduled visits for subjects or their families. This has 
proven very successful for subject retention as well. If the subject needs to be seen on the one 
day a week the clinic is closed, there is a doctor on call for research studies  or in Rajshahi or 
subjects also may come to the icddr,b or  Rajshahi Medical College Hospitals or local Rajshahi 
site clinic. 

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Unscheduled visits may occur at any time during the study.  Any of the following activities may 
be performed: 
• Targeted physical exam, if indicated.  
• Assessment of any side effects or adverse events. 
• Medical history, including any concomitant medications, reviewed. 
• If a subject complains of abdominal pain or diarrhea, a stool will be collected for ova 
and parasite to document whether Giardia or Entamoeba are present 
 
 
 
7.6 
Pregnancy Visit 
 
If a subject should become pregnant at any time during the study, she will be asked to stay in the 
study for continued evaluation. If she leaves the study before the end of her pregnancy, the study 
staff will request permission for her to be contacted at the end of her pregnancy so that she and 
her baby can be evaluated. 
 

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8 
STUDY PROCEDURES/EVALUATIONS 
 
8.1 
Clinical Evaluations 
Medical History: Complete medical history will be obtained by interview of the subjects at the 
screening visit (Visit 00B) and will be reviewed and updated on Day 1 (Visit 01) prior to the first 
study product administration.  Subjects will be queried regarding a history of significant medical 
disorders of the head, eyes, ears, nose, throat, mouth, cardiovascular system, lungs, 
gastrointestinal tract, liver, pancreas, kidney, urologic system, nervous system, blood, lymph 
nodes, endocrine system, musculoskeletal system, skin, and genital/reproductive tract.  A history 
of any allergies, cancer, immunodeficiency, psychiatric illness, substance abuse, and autoimmune 
disease will be solicited.  At follow-up visits after the first dose, an interim medical history will 
be obtained, when indicated, by interview of the subjects noting any changes since the previous 
visit.   
Concomitant Medications: Concomitant medications will include a review of all current 
medications and medications taken within 30 days prior to signing the informed consent form and 
through approximately 30 days after the first study product administration.  Prescription and over-
the-counter drugs will be included as well as vitamins and supplements.  Assessment of eligibility 
will also include a review of all permitted and prohibited medications per the Subject Inclusion 
and Exclusion Criteria (see Section 5). 
Physical Examination and Vital Signs: A physical examination limited to vital signs (blood 
pressure, pulse, temperature), observation for rashes or glossitis, and an abdominal exam will be 
performed at screening (Visit 00B). On Day 1 (Visit 01) prior to the first study product 
administration and at follow-up visits after the first administration of study product, a targeted 
physical examination will be performed, if indicated, based on subject’s interim medical history. 
Adverse Events:  Subjects will be interviewed to determine the occurrence of unsolicited AEs. 
8.2 Laboratory Evaluations 
8.2.1 Clinical Laboratory Evaluations 
Laboratory Evaluations will include: 
1. Stool examination: Stool samples will be obtained for ova and parasite testing.  
Subjects screened at icddr, b or Rajshahi Hospital will be given a stool cup for Day 1 

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sampling. Once discharged, all subjects will perform stool collections at icddr,b, 
Rajshahi Hospital, clinic or home on Days 3, 5, 7(window +1 day), 14 (window +3 
days) and 28 (window +3 days).The specimen will be kept in a cooler with an ice pack 
at the subject’s house until picked up by a Field Research Assistant and placed in a 
cooler with an ice pack for transport to the lab at icddr,b or  Rajshahi Medical College. 
Initial screening involves having a positive stool sample test for the E. histolytica or 
Giardia parasites. All laboratory personnel will be blinded, and the specimens de-
identified. 
a. Testing of stool samples will be performed by the icddr,b laboratory or 
Rajshahi Hospital  laboratory and will include: 
i. Rapid EIA and antigen detection EIA to detect E. histolytica and Giardia 
antigens to identify protozoal infections; 
ii. Wet mount for ova and parasite examination; 
iii. PVA fixation for concentration and trichrome staining for ova and parasite 
examination  
iv. Qualitative PCR to detect E. histolytica and Giardia 
a.  Off-site testing of stool samples collected at Rajshahi will be transported to icddr, 
b and shipped to UCSD to include quantitative E. histolytica or Giardia testing 
performed at UCSD on shipped aliquots of DNA extracted from positive stools at 
icddr,b.  De-identified non-infectious DNA specimens labeled with the subject code 
will be obtained from stools on Days 1, 3, 5, 7, 14, and 28 following enrollment. 
Quantitative PCR assays have been developed based on the small subunit rRNA gene, 
which is highly conserved among E. histolytica (Taniuchi, 2011) and Giardia isolates 
(Haque, 2007). 
2. 
Urine testing: 
a. Pregnancy testing at both hospital sites. Upon screening: as a condition of 
enrollment, all women of child bearing age must agree to have a pregnancy test, 
receive the results, and use effective birth control for the next four months.  Testing 
will be performed on urine using HCG test strip assays. If the test is positive or the 
woman is breastfeeding, they will not be enrolled, as auranofin is contraindicated in 
pregnant or breastfeeding women. Women who are enrolled in the study will continue 
their current effective birth control for 4 months after enrollment.  
b. Urine for proteinuria. On Visit 00B and 7: urine will be collected from all 
participants for proteinuria assessment via dipstick testing. Proteinuria testing will be 
performed at the icddr,b 
3. 
Blood testing: Blood will be drawn for a complete blood count, BUN, creatinine, ALT, and 
AST on Visit 00B and Day 7. Blood tests will be collected at the icddr,b Hospital or Rajshahi 
Hospital or Clinic and transported to the respective clinical laboratory. 

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4. 
E. histolytica and Giardia genotyping: Any stool specimen that is still positive for 
Entamoeba or Giardia cysts or trophozoites at Days 14 and 28 will have DNA genotyping 
of the parasite performed on de-identified specimens of extracted DNA. The genotypes from 
initial and final isolates will be compared to allow determination of the rate of relapse or re-
infection without linking to individual patients. The genotyping will be performed off-site 
at the University of Virginia. There will be no human DNA genotyping performed. 
8.2.2 Specimen Preparation, Handling and Shipping 
Appropriate blood and secretion precautions will be employed by all personnel in the drawing of 
blood and shipping and handling of all specimens for this study, as currently recommended by 
the Centers for Disease Control and Prevention and the National Institutes of Health. All 
potentially infectious specimens will only be transported locally to the laboratory using packing 
mandated in the Code of Federal Regulations, 42 CFR Part 72 and IATA guidelines.  
8.2.3 Instructions for Specimen Preparation, Handling, and Storage 
Any residual serum from safety screening, aliquots of stool, and parasite DNA extracted from the 
stool of enrolled subjects only will be stored in cryovials marked with the subject identification 
number and date of specimen collection at icddr,b or Rajshahi Hospital or Clinic following 
specific consent of the subjects. Long-term stored specimens will go to the Fisher Repository.  
Future studies may include measuring gold levels in the blood or stool. Other researchers may 
request access to these specimens for collaborative studies as well. Further instructions for 
specimen preparation, handling, and storage are included in the protocol-specific MOP as 
appropriate. There will be no human DNA collection or storage. 
8.2.4 Specimen Shipment 
Only noninfectious, parasite extracted DNA specimens will be shipped to UCSD for quantitative 
parasite loads or University of Virginia for genotyping. They can be sent at room temperature by 
standard international couriers, or designated members of the study team with completed 
specimen/Transport Receipt Logs. The residual long-term stored specimens will be shipped 
according to IATA guidelines to the Fisher Repository.  

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9 
ASSESSMENT OF SAFETY 
9.1 Specification of Safety Parameters 
Because this is an FDA approved drug that has been in use for more than 25 years, the potential 
AEs have been well documented in subjects in longer term therapy for rheumatoid arthritis. Of 
the AEs listed on the Insert, the most serious reactions would be an allergic reaction to gold, 
which could include anaphylactic reactions, necrotizing enterocolitis, pulmonary fibrosis, 
exfoliative dermatitis, bone marrow aplasia or other severe hematologic disorders.  
The adverse reactions incidences listed below are based on observations of 1) 4,784 Ridaura-
treated subjects in clinical trials (2,474 U.S., 2,310 foreign), of whom 2,729 were treated more 
than one year and 573 for more than three years; and 2) post-marketing experience. The highest 
incidence is during the first six months of treatment; however, reactions can occur after many 
months of therapy. With rare exceptions, all subjects were on concomitant nonsteroidal anti-
inflammatory therapy; some of them were also taking low dosages of corticosteroids. Reactions 
occurring in more than1% of Ridaura-treated patients: 
• Gastrointestinal: loose stools or diarrhea (47%); abdominal pain (14%); nausea with 
or without vomiting (10%); constipation; anorexia*; flatulence*; dyspepsia*; 
dysgeusia. 
• Dermatological: rash (24%); pruritus (17%); hair loss; urticaria. 
• Mucous Membrane: stomatitis (13%); conjunctivitis*; glossitis. 
• Hematological: anemia; leukopenia; thrombocytopenia; eosinophilia. 
• Renal: proteinuria*; hematuria. 
• Hepatic: elevated liver enzymes. 
*Reactions marked with an asterisk occurred in 3-9% of the patients. The other 
reactions listed occurred in 1-3%. Reactions occurring in less than 1% of Ridaura-
treated patients 
• Gastrointestinal: dysphagia; gastrointestinal bleeding†; melena†; positive stool for 
occult blood†; ulcerative enterocolitis. 
 

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9.2 
Methods and Timing for Assessing, Recording, and Analyzing Safety 
Parameters 
9.2.1 Adverse Events 
Adverse Event: International Conference on Harmonisation (ICH) E6 defines an AE as any 
untoward medical occurrence in a patient or clinical investigation subject administered a 
pharmaceutical product regardless of its causal relationship to the study treatment.  An AE can 
therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), 
symptom, or disease temporally associated with the use of a medicinal (investigational) product.  
The occurrence of an AE may come to the attention of study personnel during study visits and 
interviews of a study recipient presenting for medical care, or upon review by a study monitor. 
AEs not meeting the protocol-defined criteria for SAEs will be captured on the appropriate data 
collection form and electronic case report form (eCRF).  Information to be collected for 
unsolicited AEs includes event description, date of onset, licensed clinician’s assessment of 
severity and relationship to study product and alternate etiology (if not related to study product) 
(assessed only by those with the training and authority to make a diagnosis and listed on the Form 
FDA 1572 as the site principal investigator or sub-investigator), date of resolution of the event, 
seriousness and outcome.  AEs while on study will be documented appropriately regardless of 
relationship.  AEs will be followed to resolution. 
Any medical condition that is present at the time that the subject is screened will be considered 
as baseline and not reported as an AE.  However, if the severity of any pre-existing medical 
condition increases, it will be recorded as an AE. 
AEs must be graded for severity and assessed for relationship to study product (see definitions 
below).  Adverse events characterized as intermittent require documentation of onset and duration 
of each episode.  The start and stop date of each reported AE will be recorded on the appropriate 
data collection form and eCRF. 
FDA defines an AE as any untoward medical occurrence associated with the use of a drug in 
humans, whether or not considered drug related. 
Severity of Event: AEs will be assessed by a licensed study physician listed on the Form FDA 
1572 as the site principal investigator or appropriate sub-investigator using a protocol-defined 
grading system (see Section 9.2.2 and 9.2.3 ).  For events not included in the protocol-defined 
grading system, the following guidelines will be used to quantify severity: 

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• Mild (Grade 1):  Events require minimal or no treatment and do not interfere with the 
subject’s daily activities. 
• Moderate (Grade 2):  Events result in a low level of inconvenience or concern with 
therapeutic measures.  Moderate events may cause some interference with functioning and 
daily activities. 
• Severe (Grade 3):  Events that prevent the subject’s usual daily activities and may require 
systemic drug therapy or other treatment.  Severe events are usually incapacitating. 
The severity of systemic and clinical laboratory adverse events will be graded according to 
Section 9.2.2 and the toxicity tables included in the Appendices to this protocol. 
Changes in the severity of any AE will be documented to allow an assessment of the duration of 
the event at each level of intensity.  When AEs are intermittent, the onset and duration of each 
episode will be documented. 
Relationship to Study Products: The study physician’s assessment of an AE's relationship to 
study product is part of the documentation process, but it is not a factor in determining what is or 
is not reported in the study.  If there is any doubt as to whether a clinical observation is an AE, 
the event should be reported.  The relationship to study product must be assessed for AEs using 
the terms:  related or not related.  In a clinical trial, the study product must always be suspect.  To 
help assess, the following guidelines are used: 
• Related – There is a reasonable possibility that the study product caused the adverse event. 
Reasonable possibility means that there is evidence to suggest a causal relationship between 
the study product and the adverse event. 
• Not Related – There is not a reasonable possibility that the administration of the study product 
caused the event. 
9.2.2 Solicited Events 
Solicited events are AEs that are known to occur with this study product. Solicited AEs will not 
be collected that are related to any prescribed metronidazole. The below Toxicity Grading Scales 
will be used to grade events. 
Adverse event data will be collected at each visit using specific questions and/or targeted physical 
examination. Based on the adverse reactions listed on the package insert, expected side effects 
include: loose stools or diarrhea, abdominal pain, nausea with or without vomiting, constipation, 
anorexia, flatulence, dyspepsia, dysgeusia, pruritus, hair loss, urticarial, stomatitis, conjunctivitis, 

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44 
glossitis, and hematuria. Diarrhea, however, will not be recorded as a side effect because all 
participants will enroll with diarrhea, and because its resolution is the endpoint of the study. 
Physical examinations will look for abdominal pain, hair loss, urticaria, rashes, stomatitis, 
conjunctivitis, and glossitis. Hematologic, renal, or hepatic laboratory abnormalities will be 
picked up on testing at Day 7. Adverse event collection will continue until Day 28 (following 
three half-lives of the drug).  
 
Event 
Mild (Grade 1) 
Moderate (Grade 2) 
Severe (Grade 3) 
Loose stools or diarrhea 
Does not interfere with 
daily activity 
Interferes with daily 
activity 
Prevents daily activity 
(incapacitating) 
Abdominal Pain 
Does not interfere with 
daily activity 
Interferes with daily 
activity 
Prevents daily activity 
(incapacitating) 
Nausea with or without 
vomiting 
Does not interfere with 
daily activity 
Interferes with daily 
activity 
Prevents daily activity 
(incapacitating) 
Constipation 
Does not interfere with 
daily activity 
Interferes with daily 
activity 
Prevents daily activity 
(incapacitating) 
Anorexia 
Does not interfere with 
daily activity 
Interferes with daily 
activity 
Prevents daily activity 
(incapacitating) 
Flatulence 
Does not interfere with 
daily activity 
Interferes with daily 
activity 
Prevents daily activity 
(incapacitating) 
Dyspepsia 
Does not interfere with 
daily activity 
Interferes with daily 
activity 
Prevents daily activity 
(incapacitating) 
Dysgeusia 
Does not interfere with 
daily activity 
Interferes with daily 
activity 
Prevents daily activity 
(incapacitating) 
Pruritus 
Does not interfere with 
daily activity 
Interferes with daily 
activity 
Prevents daily activity 
(incapacitating) 
Hair loss 
Does not interfere with 
daily activity 
Interferes with daily 
activity 
Prevents daily activity 
(incapacitating) 
Urticaria 
Does not interfere with 
daily activity 
Interferes with daily 
activity 
Prevents daily activity 
(incapacitating) 
Stomatitis 
Does not interfere with 
daily activity 
Interferes with daily 
activity 
Prevents daily activity 
(incapacitating) 
Conjunctivitis 
Does not interfere with 
daily activity 
Interferes with daily 
activity 
Prevents daily activity 
(incapacitating) 
Glossitis 
Does not interfere with 
daily activity 
Interferes with daily 
activity 
Prevents daily activity 
(incapacitating) 

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Event 
Mild (Grade 1) 
Moderate (Grade 2) 
Severe (Grade 3) 
Hematuria 
Does not interfere with 
daily activity 
Interferes with daily 
activity 
Prevents daily activity 
(incapacitating) 
Rash 
Does not interfere with 
daily activity 
Interferes with daily 
activity 
Prevents daily activity 
(incapacitating) 
 
9.2.3 Additional Adverse Event Severity Grading 
 
Vital signs will be graded as follows: 
 
Vital Signs* 
Vital Signs 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Fever (°C) ** 
 
37.8-38.4 
 
38.5-38.9 
 
>= 39.0 
 
Hypertension 
(systolic) mm Hg 
141-150 
151-155 
>155 or  
an ER visit or hospitalization 
for malignant hypertension 
Hypertension 
(diastolic) mm Hg 
91-95 
96-100 
>100 or  
an ER visit or hospitalization 
for malignant hypertension 
Hypotension 
(systolic) mm Hg 
85-89 
80-84 
<80 or  
an ER visit or hospitalization 
for hypotensive shock 
Bradycardia – beats 
per minute *** 
50-54 
or>10 bpm less than 
baseline if baseline <60 
45-49 
or>15 bpm less than 
baseline if baseline <60 
<45 
or> 20 BPM or less than 
baseline if baseline <60, 
or an ER visit or hospitalization 
for arrhythmia 
Tachycardia – beats 
per minute 
101-115 
116-130 
>130 
or an ER visit or hospitalization 
for arrhythmia 
* Subjects should be at rest for at least 15 minutes prior to vital sign measurements 
** Oral temperature; no recent hot or cold beverages or smoking 
*** When resting heart rate is between 60-100 beats per minute. Pulse and Blood Pressure assessed at Visit 1 (Day 1) will be 
considered baseline.  Sinus bradycardia among some healthy subject populations, for example, conditioned athletes maybe 
acceptable.   
 
Abnormal laboratory findings that will trigger an Adverse Event report will include: 
• 
Hematologic: WBC <3.5 or>13.0 (109/L), Hemoglobin <11.0 or >18.0.gm/dL, Platelets 
<130 or >550 (109/L) 

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46 
• 
Hepatic: SGOT (AST) or SGPT (ALT)> 70.0 
• 
Renal: Creatinine>133 umol/L, Blood Urea Nitrogen >30 mg/dL, > 1+ proteinuria 
 
Laboratory values that are outside of the normal range, but do not meet protocol defined criteria 
as adverse events will be evaluated by a clinician for clinical significance, recorded in the 
source document, and reported as laboratory AEs if clinically significant. Other laboratory 
parameters not specified in the protocol, but performed as part of the CBC and complete 
metabolic panel, need to be evaluated by a clinician, recorded in the source document, and 
reported as laboratory AEs if clinically significant. 
 
Hematology 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
WBC 109/L (Decrease) 
3.0-3.4 
2.0-2.9 
<2.0 
WBC 109/L (Increase) 
13.1-13.9 
14.0-15.0  
>15.0  
Hgb g/dL (Decrease)  
9.1-10.9 
7.5-9.0 
<7.5 
Hgb g/dL (Increase) 
18.1-18.5 
18.6-19.0 
>19.0 
Platelets 109/L (Decrease) 
100-130 
50-99 
<50 
Platelets 109/L (Increase) 
551-600 
601-700 
>700 
 
 
 
 
 
Chemistry 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
BUN mg/dL 
31-60 
>60- and <70 
>70 
Creatinine umol/L1 (Increase)  
134-176  
177-221  
>221 
AST IU/L (Increase)  
70.1-200 
201-300 
>300 
ALT IU/L (Increase)  
70.1-200 
201-300 
>300 
 
 
Urinalysis 
Mild 
(Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Protein 
1+ 
2+ 
hospitalization or 
dialysis 
1Conversion factor from mg/dL: http://onlinelibrary.wiley.com/doi/10.1002/97808138188825.app3/pdf 
2Trace protein at screening (Visit 00B) is acceptable for inclusion into the study 

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9.2.4 Serious Adverse Events 
An adverse event or suspected adverse reaction is considered “serious” if, in the view of either 
the investigator or sponsor, it results in any of the following outcomes:  
• Death, 
• a life-threatening adverse event*,  
• inpatient hospitalization or prolongation of existing hospitalization,  
• a persistent or significant incapacity or substantial disruption of the ability to conduct normal 
life functions, or  
• a congenital anomaly/birth defect.   
• Important medical events that may not result in death, be life-threatening, or require 
hospitalizations may be considered serious when, based upon appropriate medical judgment 
they may jeopardize the patient or subject and may require medical or surgical intervention 
to prevent one of the outcomes listed in this definition.  Examples of such medical events 
include allergic bronchospasm requiring intensive treatment in an emergency room or at 
home, blood dyscrasias or convulsions that do not result in inpatient hospitalization, or the 
development of drug dependency or drug abuse. 
* Life-threatening adverse event. An adverse event is considered “life-threatening” if, in the view 
of either the investigator or sponsor, its occurrence places the patient or subject at immediate risk 
of death.  It does not include an adverse event, had it occurred in a more severe form, might have 
caused death. 
All SAEs will be: 
• recorded on the appropriate AE eCRF 
• followed through resolution by a study clinician 
• reviewed and evaluated by a study clinician 
9.2.5 Procedures to be Followed in the Event of Abnormal Laboratory 
Test Values or Abnormal Clinical Findings 
The site principal investigator or appropriate co-investigator is responsible for reporting all 
AE/SAEs that are observed or reported during the study, regardless of the relationship to study 
product.  AE/SAEs, abnormal clinical laboratory test values, or abnormal clinical findings will 

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48 
be documented, reported, and followed until levels return to baseline or until the abnormal value 
is determined to be not clinically significant. 
9.3 Reporting Procedures 
9.3.1 Serious Adverse Events 
SAEs will be followed until resolution even if this extends beyond the study-reporting period.  
Resolution of an SAE is defined as the return to pretreatment status or stabilization of the 
condition with the expectation that it will remain chronic.  
Any AE that meets a protocol-defined serious criterion must be submitted immediately 
(within 24 hours of site awareness) on an SAE form to the DMID Pharmacovigilance Group, 
at the following address:  
DMID Pharmacovigilance Group 
Clinical Research Operations and Management Support (CROMS) 
6500 Rock Spring Dr. Suite 650 
Bethesda, MD 20814, USA 
SAE Hot Line: 1-800-537-9979 (US) or 1-301-897-1709 (outside US) 
SAE FAX Phone Number: 1-800-275-7619 (US) or 1-301-897-1710 (outside US) 
SAE Email Address: PVG@dmidcroms.com 
In addition to the SAE form, selected SAE data fields must also be entered into Emmes Advantage 
eClinical system. Please see the protocol-specific MOP for details regarding this procedure. 
Other supporting documentation of the event may be requested by the DMID Pharmacovigilance 
Group and should be provided as soon as possible. 
The site will notify the ISM when an SAE is provided to the DMID Pharmacovigilance Group.  
The DMID Medical Monitor and DMID Clinical Project Manager will be notified of the SAE by 
the DMID Pharmacovigilance Group.  The DMID Medical Monitor will review and assess the 
SAE for regulatory reporting and potential impact on study subject safety and protocol conduct. 
At any time after completion of the study, if the site principal investigator or appropriate sub-
investigator becomes aware of an SAE that is suspected to be related to study product, the site 
principal investigator or appropriate sub-investigator will report the event to the DMID 
Pharmacovigilance Group. 

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9.3.2 Regulatory Reporting for Studies Conducted Under 
DMID-Sponsored IND 
Following notification from the investigator, DMID, the IND sponsor, will report any suspected 
adverse reaction that is both serious and unexpected.  DMID will report an adverse event as a 
suspected adverse reaction only if there is evidence to suggest a causal relationship between the 
drug and the adverse event.  DMID will notify FDA and all participating investigators (i.e., all 
investigators to whom the sponsor is providing drug under its INDs or under any investigator’s 
IND) in an IND safety report of potential serious risks from clinical trials or any other source, as 
soon as possible, but in no case later than 15 calendar days after the sponsor determines that the 
information qualifies for reporting as specified in 21 CFR Part 312.32. DMID will also notify 
FDA of any unexpected fatal or life-threatening suspected adverse reaction as soon as possible 
but in no case later than 7 calendar days after the sponsor’s initial receipt of the information. 
Relevant follow up information to an IND safety report will be submitted as soon as the 
information is available.  Upon request from FDA, DMID will submit to FDA any additional data 
or information that the agency deems necessary, as soon as possible, but in no case later than 15 
calendar days after receiving the request. 
All serious events designated as “not related” to study product(s), will be reported to the FDA at 
least annually in a summary format. 
9.3.3 Reporting of Pregnancy 
Pregnant women are not eligible to participate in the study. Women are counseled regarding 
prevention of pregnancy and encouraged to make every effort to avoid pregnancy during study 
participation.  If a study subject becomes pregnant during study participation, no further doses of 
study agent will be given. The basic information about the pregnancy is recorded on the 
“Pregnancy” case report form via the Emmes Advantage eClinical system. If there are 
complications during the pregnancy, the complications are recorded as adverse events in the usual 
way. The subject is asked to report outcome of the pregnancy. If there is a congenital anomaly in 
the infant, this is recorded as a serious adverse event (SAE) in the data forms for the mother (i.e., 
the study subject). Efforts will be made to follow all pregnancies reported during the course of 
the study to pregnancy outcome pending the subject’s permission. 
9.4 Type and Duration of the Follow-up of Participants after Adverse Events 
AEs and SAEs will be followed from the time of the first receipt of study drug Day 1 (Visit 01) 
through 28 days after receipt of study product. 
AEs and SAEs will be followed until resolution even if this extends beyond the study-reporting 
period (3 months after the final study visit).  Resolution of an AE/SAE is defined as the return to 

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50 
pretreatment status or stabilization of the condition with the expectation that it will remain 
chronic. 
If the site principal investigator or appropriate sub-investigator becomes aware of a sign or 
symptom and the site principal investigator or appropriate sub-investigator decides to bring the 
subject in for an evaluation to determine etiology, then the site principal investigator or 
appropriate sub-investigator, at their own discretion, can determine what further testing is 
appropriate. 
Follow-up procedures, evaluations, and outcomes will be recorded on the appropriate data 
collection form. 
9.4.1. Halting Rules 
Individual Halting Rules: Dosing for an individual subject will be discontinued if they develop 
any grade 3 drug-related event. The DSMB has requested to be notified of any individual subject 
who experiences a grade 3 drug-related event, however, the study will continue enrollment. 
Study Halting Rules: Enrollment, dosing and study procedures will be halted for DSMB 
review/recommendation if any of the following are reported: 
• Any death occurring after administration of study drug through the subject’s last study 
visit that was not the result of trauma or accident, regardless of relatedness to study 
product. 
• Any subject experiences a study drug-related SAE from the time of receipt of study drug 
through the subject’s last study visit after relatedness is finalized by DMID. 
• Occurrence of a severe allergic/hypersensitivity reaction (anaphylaxis) within 48 hours of 
dosing, requiring hemodynamic support with vasoactive medications or mechanical 
ventilation; the signs/symptoms will include any of the following: bronchospasm, 
dyspnea, wheezing, stridor, hypoxemia urticaria, angioedema, hives, and facial or 
oropharyngeal edema. 
 
If any of the halting rules are met following any subject receipt of study drug, the study will not 
continue with the remaining enrollments without a review by and recommendation from the 
DSMB to proceed. 
DMID 
retains 
the 
authority 
to 
suspend 
additional 
enrollment 
and 
study 
interventions/administration of study product during the entire study, as applicable. 

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9.4.2. Safety Oversight (DSMB) 
9.4.2 Independent Safety Monitor (ISM) 
The ISM is a physician with relevant expertise whose primary responsibility is to provide 
independent safety monitoring in a timely fashion.  For this study an ISM is not required. 
However, at each site, and at the request of DMID, in real time, the local PI should be able to 
identify an independent physician to function as ad-hoc ISM. That person should have the 
privileges to examine the subject, review the subject medical and study record and provide an 
independent medical assessment and recommendation to DMID. 
9.4.3 Data Safety Monitoring Board (DSMB) 
Safety oversight will be conducted by a DSMB that is an independent group of experts that 
monitors subject safety and advises DMID. The DSMB members will be separate and 
independent of study personnel participating in this trial and should not have scientific, financial 
or other conflict of interest related to the study. The DSMB will consist of members with 
appropriate expertise to contribute to the interpretation of the data from this trial. The membership 
will include a chairperson and a statistician who are experienced in clinical trials conduct and 
have prior DSMB experience. The DSMB will review study progress and clinical and safety data 
at the following time points: 
• Data review for safety at study specific time frames; at least annually. 
• Data review for efficacy will be performed at the completion of enrollment. No 
interim analysis is planned.  
• Study enrollment will be stopped for DSMB review of safety data when the study 
reaches 50% enrollment (i.e. 68 subjects enrolled).  DSMB will review safety data 
after the 68th subject completed Visit 8. 
• Final review meeting: 6 to 8 months after clinical database lock to review the 
cumulative unblinded safety and efficacy data for the study.  The data will be 
provided in a standard summary format.  The DSMB may be asked to provide 
recommendations in response to questions posed by DMID. 
• Ad hoc review: may be in to an anticipated safety issue such as a halting rule being 
met. 
• Additionally there will be ongoing DSMB notification of Grade 3 drug related 
events, after which the DSMB will then decide if an Ad Hoc DSMB meeting will be 
required 
The DSMB will operate under the rules of a DMID-approved charter that will be written at the 
organizational meeting of the DSMB. At this time, each data element that the DSMB needs to 
assess will be clearly defined. Procedures for DSMB reviews/meetings will be defined in the 
charter. The DSMB will review applicable data to include, but not limited to, study progress and 
participant clinical and safety data which may include enrollment and demographic information, 

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medical history, concomitant medications, physical assessments, clinical laboratory values, 
dosing, AE/SAEs, protocol defined SAEs, and assays results. Additional data may be requested 
by the DSMB, and interim statistical reports may be generated as deemed necessary and 
appropriate by DMID. The DSMB may receive data in aggregate and presented by group. The 
DSMB will meet and review this data at scheduled time points or ad hoc as needed during the 
study as defined in the protocol and DSMB charter. As an outcome of each review/meeting, the 
DSMB will make a recommendation as to the advisability of proceeding with the study (as 
applicable), and to continue, modify, or terminate the study.  
 
DMID or the DSMB chair may convene the DSMB on an ad hoc basis according to protocol 
criteria or if there are immediate concerns regarding observations during the course of the study. 
The DMID Medical Monitor is empowered to stop enrollment and dosing of the study drug if 
halting criteria are reported. The DMID Medical Monitor will be responsible for reviewing any 
protocol defined SAEs in real time. The DSMB will review SAEs on a regular basis and ad hoc 
during the study. 
 

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10. CLINICAL MONITORING STRUCTURE 
10.1 Site Monitoring Plan 
Site monitoring is conducted to ensure that the human subject protections, study and laboratory 
procedures, study intervention administration, and data collection processes are of high quality 
and meet sponsor, ICH/GCP guidelines and applicable regulations, and that the study is conducted 
in accordance with the protocol, protocol-specific MOP and applicable sponsor standard 
operating procedures.  DMID, the sponsoring agency, or its designee will conduct site-monitoring 
visits as detailed in the clinical monitoring plan. 
Site visits will be made at standard intervals as defined by DMID and may be made more 
frequently as directed by DMID.  Monitoring visits will include, but are not limited to, review of 
regulatory files, accountability records, eCRFs, informed consent forms, medical and laboratory 
reports, and protocol and GCP compliance.  Site monitors will have access to the study site, study 
personnel, and all study documentation according to the DMID-approved site monitoring plan.  
Study monitors will meet with site principal investigators to discuss any problems and actions to 
be taken and document visit findings and discussions. 

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11. STATISTICAL CONSIDERATIONS 
11.1 Study Hypotheses 
This proposed clinical trial will test the hypothesis that oral auranofin is effective for the 
treatment of amebiasis and giardiasis, separately. Because this trial will re-profile an FDA-
approved drug that is still in use, we propose a Phase IIa, randomized, single-blinded, placebo-
controlled superiority clinical trial comparing auranofin (standard dose of 6 mg daily) for 7 days 
for treatment of amebiasis or 5 days for giardiasis to placebo in adults. The study will have two 
primary outcomes that will be tested on two different sets of 68 subjects (i.e. one set that has 
amebiasis and the other set has giardiasis).  The amebiasis primary outcome will be the 
proportion of subjects with resolution of diarrhea (less than 3 loose stools/ 24 hours) by Day 7. 
The giardiasis primary outcome will be the proportion of subjects and resolution of diarrhea (less 
than 3 loose stools/24 hours) by Day 5. Additional secondary outcomes with E. histolytica 
include the proportion of subjects with positive EIA for E. histolytica and trophozoites on smear 
at enrollment with parasitological response of amebiasis (as determined by no detection of 
trophozoites of E. histolytica on microscopic exam day 3, 5, and 7; the rate of decrease of 
trophozoite/cyst load by qPCR in stools by Days 3, 5, and 7; the proportion of subjects with a 
negative stool antigen test by Days 3, 5, 7, and 14; the proportion of subjects with sustained cure 
(no detection of trophozoites by microscopic exam) at 14 and 28 days; the proportion of subjects 
with relapse (same strain) or re-infection (new strain) with positive stools at 14 and 28 days by 
genotyping the initial vs. subsequent strain. For patients with giardiasis, secondary outcomes 
include the proportion of subjects with positive EIA for Giardia and trophozoites on smear at 
enrollment with parasitological response (no detection of trophozoites on microscopic exam) by 
Days 3 and 5; the rate of decrease of trophozoite/cyst load by qPCR in stools by Days 3 and 5; 
the proportion of subjects with negative stool antigens by Days 3, 5, and 7; the proportion of 
subjects with sustained cure (no detection of trophozoites by microscopic exam) at 14 and 28 
days; the proportion of subjects with relapse (same strain) or re-infection (new strain) with 
positive stools at Days 14 and 28 by genotyping the initial vs. subsequent strain.  
11.2 Sample Size Considerations 
The primary amebiasis outcome for this study is to compare the proportion of subjects with 
resolution of diarrhea (less than 3 loose stools/ 24 hours) by Day 7 of therapy, in which placebo 
is expected to have a clearance rate of approximately 15-20%.  Sample size calculations for both 
primary endpoints are based on a two-sample, two-sided binomial test for proportions, with alpha 
set to 0.05.  Calculations are performed using the R statistical package (Version 3.0.2; 
http://www.r-project.org).  The primary amebiasis endpoint in this aim is to compare the 
resolution of diarrhea between the two study arms to demonstrate that auranofin increases 

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55 
resolution of diarrhea. For this power analysis, we assume the following: a predicted diarrhea 
resolution rate of at least 80% (based on treatment results with current standard therapy with 
metronidazole, Pherson, 1984) in the auranofin treatment group.   
With these assumptions, assuming 10% attrition (determined from the average of multiple other 
clinical trials in the same districts), a sample size of 68 with amebiasis (34 per arm; 60 completers 
in total) achieves greater than 95% power to detect a difference between the group proportions of 
60%. The high statistical power for the primary endpoint is planned to also ensure adequate power 
for the secondary endpoints.  
For the primary giardiasis endpoint of proportion of subjects with resolution of diarrhea by Day 
5, a similar sample size of 34 per arm will achieve 85% power to detect a 40% difference between 
treatment arms with a predicted clearance rate of 70% (based on response rates to metronidazole 
or tinidazole, Gardiner, 2001) and a 30% clearance rates in placebo. 
11.3 Planned Interim Analyses 
There will be no planned interim analyses for efficacy or futility conducted for this study, but the 
DSMB may modify this during ongoing safety monitoring. The study team will review all adverse 
events by cumulative reports on a monthly basis.  Adverse events will be graded using the 
protocol defined grading system. The DSMB, established by the study team, will monitor this 
study. 
 
In the absence of any SAEs, the DSMB may review the safety data when the study reaches 50% 
enrollment (i.e. 34 subjects enrolled per parasite group). This review will include safety data after 
the 68th subject completes Visit 08.  
11.3.1 Safety Review 
Interim safety review may include enrollment and demographic information, medical history, 
concomitant medications, physical assessments, clinical laboratory values, dosing, and solicited 
and unsolicited AE/SAEs.  Additional data may be requested by the DSMB, and interim statistical 
reports may be generated as deemed necessary and appropriate by DMID.  The DSMB may 
receive data in aggregate and presented by group. The DSMB will meet and review this data at 
scheduled time points or ad hoc as needed during the study as defined in the DSMB charter.  As 
an outcome of each review/meeting, the DSMB will make a recommendation as to the advisability 
of proceeding with study vaccinations (as applicable), and to continue, modify, or terminate the 
study. 

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Additionally, the study will be monitored to determine if any of the halting rules described in 
Section 9.5 are met. 
11.4 Final Analysis Plan 
Analysis Plan for Primary Outcomes. The primary amebiasis endpoint is resolution of diarrhea 
by Day 7. Parasite clearance rates between the placebo and auranofin arms for Entamoeba will 
be compared using a Fisher’s exact test for proportions. Differences in the rates between the two 
groups, along with the odds ratio (OR) and their 95% confidence intervals will be reported.  As a 
secondary analysis, multivariable logistic regression analysis will be performed to study the 
association between clearance rates and intervention arm, adjusting for baseline demographic, 
stratification variables, and clinical characteristics. Variables significantly associated with both 
treatment group and outcome (p<0.10) will be included in a multivariable logistic regression 
model as covariates.   There will be no planned interim analyses for efficacy or futility conducted 
for this study, but the DSMB may modify this during ongoing safety monitoring.  
 
Similarly, for the giardiasis primary endpoint, methods analogous to the amebiasis primary 
endpoint will be used to determine the resolution of diarrhea by Day 5. 
 
Analysis Plan for Secondary Outcomes. The secondary outcome measures are: Entamoeba: 
Proportion of subjects with positive EIA for E. histolytica and positive smear for trophozoites on 
enrollment  with parasitological response (no detection of trophozoites on microscopic exam) by 
Day 7; proportion of subjects with positive EIA for E. histolytica and positive smear for 
trophozoites on enrollment with parasitological response (no detection of trophozoites on 
microscopic exam or negative antigen detection) by Day  3 and 5; rate of decrease of 
trophozoite/cyst load by qPCR in stools by Days 3, 5 and 7; proportion of subjects with negative 
stool antigen test by Days 3, 5, 7, and 14;  proportion of subjects with sustained cure (no detection 
of trophozoites by microscopic exam) at 14 and 28 days; and the proportion of subjects with 
relapse (same strain) or re-infection (new strain) with positive stools at Days 14 and 28 by 
genotyping the initial vs. subsequent strain, and time to resolution of diarrhea.  
 
Giardia: Proportion of subjects with positive EIA for Giardia and positive smear for trophozoites 
on enrollment with parasitological response (no detection of trophozoites on microscopic exam) 
by Day 3 and 5; rate of decrease of trophozoite/cyst load by qPCR in stools by Days 3 and 5; 
proportion of subjects with negative stool antigens by days 3 and ; proportion of subjects with 
sustained cure (no detection of trophozoites by microscopic exam) at 14 and 28 days; the 
proportion of subjects with relapse (same strain) or re-infection (new strain) with positive stools 
at Days 14 and 28 by genotyping the initial vs. subsequent strain and time to resolution of diarrhea.  
 

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57 
Methods analogous to the analysis of the primary outcomes, such as Fisher’s exact test, will be 
applied for categorical secondary outcomes (proportion of trophozoite/cyst load in stools from 
enrollment by quantitative PCR at days 3, 5, and 7; and percentage of subjects who have relapsed 
at 14 and at 28 days).  For continuous outcomes, such as count data, t-tests or equivalent non-
parametric alternatives will be considered.  No adjustments will be made for multiple testing of 
secondary outcomes.  Longitudinal modeling, via mixed effects models, will be considered to 
compare the rate of change in loads between the two study arms.  Appropriate treatment by time 
interaction terms will be included. Kaplan-Meier curves and log rank tests will be used to compare 
time to diarrhea resolution between the treatment and control arm for each disease group.   
 
In general, analyses will incorporate the modified intent-to-treat (mITT) principle. Participants 
who are “randomized and receive at least one dose of study medication” will be included in the 
mITT population. Subjects who terminate their treatment regimen and receive metronidazole due 
to worsening symptoms will be imputed as treatment failures for the primary outcome.  That is, 
they will be imputed as still symptomatic by Day 7 in the amebiasis group, and by Day 5 in the 
giardiasis group.  Secondary outcomes measured at Days 3, 5, and 7 will be imputed analogously, 
with the exception of the quantitative trophozoite/cyst load. Multiple imputation may be used to 
adjust for missing data in the primary and secondary analyses on the mITT population.  All results 
will be reported as point estimates (odds ratios or mean differences across groups, as appropriate) 
and interval estimates (95% confidence intervals).  All tests of significance for the secondary 
outcomes will be 2-sided and no adjustments will be made for multiple comparisons.  A p-value 
of 0.05 will be considered statistically significant.  The Statistical Analysis Plan (SAP) will 
provide further details. In case the language in this section differs from the language in the SAP, 
the SAP takes precedence. 
In the event that recruitment and follow-up for either the Giardiasis arm or the Amebiasis arm is 
completed ahead of the other, the primary and secondary endpoints may be analyzed for that arm 
using the specified methods.  This report will be considered the interim CSR and will contain 
detailed unblinded information about the participants, their immunological response to treatment 
as well as their side effects and any laboratory abnormalities.  The interim CSR will be followed 
with a release of data corresponding to the report.  In order to maintain the blind and allow 
appropriate follow up of the remaining arm, no endpoint data for the remaining arm will be 
included in the interim CSR and no data related to that arm will be released.  An amendment to 
the interim CSR will provide primary and secondary analysis of the remaining arm when all 
primary and secondary endpoint data are available for the second arm.  The official reports will 
be drafted by Emmes and reviewed by the PI and DMID prior to finalization.  Additional 
information regarding the methods will be provided in the study SAP.     
The purpose of this study is to assess the efficacy of auranofin (Ridaura) for the treatment of 
amebiasis and giardiasis and the timely release of this information, once available, will support 
improved clinical care.  It is anticipated that the results of this study will be presented to the 

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scientific community via oral presentations at meetings and written publications in scientific 
journals following finalization of the interim CSR. The data to be presented and the authorship 
will be discussed between partners before any official communication. 

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12 
SOURCE DOCUMENTS AND ACCESS TO SOURCE 
DATA/DOCUMENTS 
The site will maintain appropriate medical and research records for this trial, in compliance with 
ICH E6, Section 4.9, and regulatory and institutional requirements for the protection of 
confidentiality of subjects.  The site will permit authorized representatives of the DMID, its 
designees, and appropriate regulatory agencies to examine (and when required by applicable law, 
to copy) clinical study records for the purposes of quality assurance reviews, audits, monitoring 
and evaluation of the study safety and progress.  These representatives will be permitted access 
to all source data, which include, but are not limited to, hospital records, clinical and office charts, 
laboratory notes, memoranda, evaluation checklists, pharmacy dispensing records, recorded data 
from automated instruments, copies or transcriptions certified after verification as being accurate 
and complete, microfiches, photographic negatives, microfilm or magnetic media, x-rays, and 
subject files and records kept at the pharmacy, at the laboratories, and medico-technical 
departments involved in the clinical trial.  Source data are all information, original records of 
clinical findings, observations, or other activities in a clinical trial necessary for the reconstruction 
and evaluation of the trial.  Data collection forms will be derived from the eCRFs and be provided 
by the SDCC. 
 
 

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13 
QUALITY CONTROL AND QUALITY ASSURANCE 
Following a written DMID-accepted site clinical quality management plan (CQMP), the 
investigational site is responsible for conducting routine quality assurance and quality control 
activities to internally monitor study progress and protocol compliance.  
 
The PI will provide direct access to all trial-related sites, source data/documents, and reports for 
the purpose of monitoring and auditing by the sponsor, and inspection by local and regulatory 
authorities. The PI will ensure all study personnel are appropriately trained and applicable 
documentation is maintained on site. Staff Training will include Institution-specific Training, 
Protocol-specific Training, and DMID-specific training.  
 
DMID-designated clinical monitors will verify that the clinical trial is conducted and data are 
generated, documented (recorded), and reported in compliance with the protocol, ICH/GCP 
guidelines, and the applicable regulatory requirements. Clinical monitoring reports will be 
submitted to DMID. 
The Statistical and Data Coordinating Center will implement quality control procedures 
beginning with the data entry system and generate data quality control checks that will be run on 
the database. Any missing data or data anomalies will be communicated to the site(s) for 
clarification and resolution. 
 

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14 ETHICS/PROTECTION OF HUMAN SUBJECTS 
14.1 Ethical Standard 
The principal investigator will ensure that this trial is conducted in full conformity with principles 
of the Belmont Report:  Ethical Principles and Guidelines for the Protection of Human Subjects 
of Research of the National Commission for the Protection of Human Subjects of Biomedical and 
Behavioral Research (April 18, 1979) and codified in 45 CFR 46, 21 CFR 50 and 56, and ICH 
E6; 62 Federal Regulations 25691 (1997), if applicable.  The site principal investigator’s 
Institution will hold a current Federal Wide Assurance (FWA) issued by the Office of Human 
Research Protection (OHRP) for federally funded research. 
14.2 Institutional Review Board/Ethics Committee 
UCSD is the lead institution in this project and where the PI is located. Quantitative PCR testing 
of positive stool specimens will be performed at UCSD. Although no direct human subjects 
research will occur at this institution, UCSD will be responsible for all regulatory matters in 
conjunction with our collaborators. This includes coordination of all IRB approvals, quality 
assurance, data safety and monitoring and protocol development. The International Centre for 
Diarrhoeal Research, Bangladesh (icddr,b) and  Rajshahi Medical College Hospital and Clinic  
are the  sites where human subjects research procedures will be performed, with the option for 
home visits also conducted as indicated. This includes screening, recruiting, and collecting all 
data/samples and following up subjects as detailed in Section 7.  The icddr,b Ethics Committee 
will also review and approve study protocols which is also is used for approval of joint Rajshahi 
Medical College protocol.  Dr. Petri’s lab at the University of Virginia will perform off site 
molecular testing of specific strains of Giardia or E. histolytica to identify relapse or re-infection.  
14.3 Informed Consent Process 
Informed consent is a process that is initiated prior to the individual’s agreeing to participate in 
the study and continuing throughout the individual’s study participation.  Extensive discussion of 
risks and possible benefits of this therapy will be provided to the subjects and their families.  
Consent forms describing in detail the Study Agent, auranofin, and risks are given to the subject 
and written documentation of informed consent is required prior to starting study 
agent/intervention.  Consent forms will be IRB approved and the subject will be asked to read 
and review the document. Upon reviewing the document, the investigator will explain the 
research study to the subject and answer any questions that may arise.  The subjects will sign the 
informed consent document prior to any procedures being done specifically for the study.  The 
subjects should have sufficient opportunity to discuss the study and process the information in 

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the consent process prior to agreeing to participate.  The subjects may withdraw consent at any 
time throughout the course of the trial.  A copy of the informed consent document will be given 
to the subjects for their records.  The rights and welfare of the subjects will be protected by 
emphasizing to them that the quality of their medical care will not be adversely affected if they 
decline to participate in this study. 
The informed consent procedure is designed to maximize the potential subject’s comprehension 
of study procedures and to ensure that participation is voluntary.  Before a subject is enrolled in 
the study, the purpose, the procedures to be followed, and the risks and benefits of participation 
will be explained by the clinic staff, and signed informed consent from the study subject will be 
obtained. For those subjects unable to read or sign the consent documents, a literate witness will 
be present to sign on behalf of the subject after the subject has given oral consent. In addition to 
the signature of a literate witness, agreement of subjects who are illiterate will be indicated by 
including his/her thumbprint on the consent form, in accordance with the WHO guidelines on the 
consent 
process 
for 
illiterate 
individuals 
(http://www.who.int/rpc/research_ethics/Process_seeking_IF_ printing.pdf). 
Clinic staff will be trained to respond to the subject’s questions and concerns. Details of study 
participation, including the need for follow-up contact, will be described in both the consent form 
and explained verbally. The research staff who will be responsible for obtaining informed consent 
will assess whether the potential subject has understood critical aspects of the study and consent 
form by asking key questions (e.g., “How much time will this take you?”; “What are the possible 
risks to you?”). Errors will be corrected and these potential subjects will then be asked if they 
need further clarification. If, after further attempts to clarify any misunderstandings, we determine 
that they may not fully comprehend the critical aspects of the study, they will not be enrolled. If 
a potential subject decides he/she does not wish to participate, his/her decision will be honored 
regardless of how well they comprehend the study information. The subject may refuse to 
participate or withdraw at any time without jeopardy to the medical care they regularly receive. 
The investigator will explain to the subject that only parasite DNA samples will be collected and 
analyzed and that no human genetic material will be collected.  
A copy of the consent form, which includes a description of the study, will be provided to all 
subjects, which includes contact information of the main investigators in Dhaka and Rajshahi 
Bangladesh, and the U.S. Consent forms will be written in simple language at a fourth-grade 
reading level, and will be translated into Bengali. In the case of subjects with low or no literacy, 
the methods described above of: 1) Verbally reviewing the consent form; and 2) Giving a simple 
test about points made in each paragraph of the consent, should open enrollment to all regardless 
of literacy level.  Participants will be encouraged to ask questions and retested if they do not fully 
understand any of the study protocols. Prior to study implementation we will receive approval 
from the UCSD Institutional Review Board (IRB) Committee on Human Research, the icddr,b 

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Ethical Review Committee in Bangladesh and the UVa Institutional Review Board (IRB) for 
Health Sciences Research. 
Participation in this study is completely voluntary, and the alternative to study participation is not 
to participate. Participants may refuse to participate or withdraw at any time without jeopardy to 
the medical care they regularly receive. Treatment for diarrhea is available at health clinics in 
Bangladesh. If a subject chooses not to participate in this study or is deemed ineligible to 
participate, he/she may ask the study staff for a referral to their nearest clinic. Whether they join 
this study or not, subjects may also ask the study staff for information about infectious diseases, 
such as Giardia and E. histolytica. 
14.4 Exclusion of Women, Minorities, and Children  
(Special Populations) 
This study will be inclusive of all adults who meet the inclusion/exclusion criteria, regardless of 
religion, sex, or ethnic background. Women who are pregnant or breastfeeding, women of 
childbearing potential who do not agree to practice effective contraception, and children are 
excluded for safety reasons. Should the outcome of this study be deemed acceptable, additional 
trials may be initiated in other populations. 
14.5 Subject Confidentiality 
Subject confidentiality is strictly held in trust by the participating investigators, their staff, and 
the sponsor(s) and their agents.  This confidentiality is extended to cover testing of biological 
samples and genetic tests in addition to the clinical information relating to participating subjects. 
The study protocol, documentation, data, and all other information generated will be held in strict 
confidence.  No information concerning the study or the data will be released to any unauthorized 
third party without prior written approval of the sponsor. 
The study monitor or other authorized representatives of the sponsor may inspect all documents 
and records required to be maintained by the investigator, including but not limited to, medical 
records (office, clinic, or hospital) and pharmacy records for the subjects in this study.  The 
clinical study site will permit access to such records. 
To guard confidentiality, only the subject's identification number will appear on screening forms, 
questionnaires, databases and biological samples. The key to subjects’ identification numbers will 
be encrypted in a computer file, which will be locked in the field coordinator’s office. Only the 
site PI and field coordinator will be able to un-encrypt the computer file. Contact information for 

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the subject so that s/he can be located at follow-up visits will be stored in locked file cabinets in 
locked offices at the clinic, and will be destroyed once follow-up is completed. 
14.6 Study Discontinuation 
If the trial is discontinued, subjects who had previously signed the informed consent form, and 
were randomized and treated will continue to be followed for safety assessments.  No further 
study treatments will be administered. 
14.7 Future Use of Stored Specimens 
At the time of informed consent, the Investigator will seek permission from subjects for use of 
their remaining sample for possible future research studies, investigating parasitic disease, such 
as the effect of parasitic infections and/or auranofin on the stool microbiome. All samples will be 
stored in a freezer at the icddr,b or Rajshahi Medical College Hospital for 3-4 days maximum, 
except for those that test positive for Giardia or Entamoeba. Samples from Rajshahi Medical 
College Hospital will be sent to icddr,b first , then to UCSD/UVA. These will be stored in the 
icddr, b freezer for the entire duration of the study, after which they will be stored indefinitely at 
the Fisher Repository Samples and may be shared with other investigators at other institutions.  
The samples will not be sold or used directly for production of any commercial product.  No 
human genetic material will be collected or stored and no human genetic tests will be performed 
on any samples.  Each sample will be de-identified and encoded (labeled) only with a barcode 
and a unique tracking number to protect subject’s confidentiality. 
There are no benefits to subjects in the collection, storage and subsequent research use of 
specimens.  Reports about future research done with subject’s samples will NOT be kept in their 
health records. 
Subjects may be given the option to decide if they want their samples to be used for future research 
or have their samples destroyed at the end of the trial.  The subject’s decision can be changed at 
any time prior to the end of the trial by notifying the study doctors or nurses in writing.  However, 
if the subject originally consents to future use and subsequently changes his/her decision, any data 
from a previously collected sample may still be used for this research. 

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15 DATA HANDLING AND RECORD KEEPING 
The investigator is responsible to ensure the accuracy, completeness, legibility, and timeliness of 
the data reported.  All source documents should be completed in a neat, legible manner to ensure 
accurate interpretation of data.  Black ink is required to ensure clarity of reproduced copies.  When 
making changes or corrections, cross out the original entry with a single line, and initial and date 
the change.  DO NOT ERASE, OVERWRITE, OR USE CORRECTION FLUID OR TAPE ON 
THE ORIGINAL. 
Copies of the electronic CRF (eCRF) will be provided for use as source documents and 
maintained for recording data for each subject enrolled in the study.  Data reported in the eCRF 
derived from source documents should be consistent with the source documents or the 
discrepancies should be explained 
DMID and/or it designee will provide guidance to investigators on making corrections to the 
source documents and eCRF. 
15.1 Data Management Responsibilities 
All data collection forms and laboratory reports must be reviewed by the clinical team and data 
entry personnel, who will ensure that they are accurate and complete.  Adverse events must be 
recorded on the appropriate data collection form, assessed for severity and relationship, and 
reviewed by the site principal investigator or appropriate sub-investigator. 
Data collection is the responsibility of the study personnel at the participating sites under the 
supervision of the respective site principal investigator.  During the study, the site principal 
investigator must maintain complete and accurate documentation for the study. 
EMMES, The SDCC for this study will be responsible for data management, quality review, 
analysis, and reporting of the study data. 
15.2 Data Capture Methods 
Clinical (including, but not limited to, AE/SAEs, concomitant medications, medical history, 
physical assessments, and clinical laboratory values), data will be entered into a 21 CFR 11-
compliant Internet Data Entry System provided by the SDCC.  The data system includes password 
protection and internal quality checks, such as automatic range checks, to identify data that appear 
inconsistent, incomplete, or inaccurate.  Clinical data will be entered directly from the data 
collection forms completed by the study personnel. 

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15.3 Types of Data 
Data for this study will include safety, laboratory, and outcome measures. 
15.4 Timing/Reports 
The study team will review all adverse events by cumulative reports of the treatment arms on a 
monthly basis. 
 
Asymptomatic subjects (34) enrolled under Version 1-4 of the protocol will have their data 
reviewed and clinical database locked for analysis first and a separate report generated.  
A final report will then be prepared following the availability of all the safety and outcome data 
for one study arm with symptomatic patients (Giardiasis or Amebiasis). An amendment to the 
final report will be produced following the availability of all safety and outcome data for the 
remaining study arm.  Interim statistical reports may be generated as deemed necessary and 
appropriate by DMID.  Safety and immunogenicity summary reports may be generated for the 
DSMB. 
After full analysis and final reporting is complete, and upon request and DMID approval, the 
SDCC will provide the site with a summary of results by treatment group and/or subject treatment 
assignments.  In this regard, the participating site requesting such information to share with study 
subjects must do so in compliance with their respective IRB guidelines. 
15.5 Study Records Retention 
Study records and reports, including, but not limited to, case report forms (CRFs), source 
documents, informed consent forms (except for future use informed consent forms), laboratory 
test results, and medication inventory records, shall be retained for 2 years after a marketing 
application is approved for the drug; or, if an application is not approved for the drug, until 2 
years after shipment and delivery of the drug for investigational use is discontinued and FDA has 
been so notified.  The site must contact DMID for authorization prior to the destruction of any 
study records.  Informed consent forms for future use will be maintained as long as the sample 
exists. 
15.6 Protocol Deviations 
A protocol deviation is any noncompliance with the clinical trial protocol, Good Clinical Practice 
(GCP), or Manual of Procedures requirements.  The noncompliance may be either on the part of 
the subject, the investigator, or the study site staff.  As a result of deviations, corrective actions 
are to be developed by the site and implemented promptly. The site will use continuous vigilance 

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to identify and report deviations according to the guidelines of the IND sponsor, if applicable. 
Protocol deviations will be sent to the local IRB/IEC per their guidelines.   
 
These practices are consistent with ICH E6: 
4.5 Compliance with Protocol, Sections 4.5.1, 4.5.2, and 4.5.3 
5.1 Quality Assurance and Quality Control, Section 5.1.1 
5.20 Noncompliance, Sections 5.20.1, and 5.20.2 
It is the responsibility of the site principal investigator and other study personnel to use continuous 
vigilance to identify and report deviations within five working days of identification of the 
protocol deviation, or within five working days of the scheduled protocol-required activity.  All 
deviations must be promptly reported to DMID, via the SDCC’s Emmes Advantage eClinical  
system.  
All protocol deviations, as defined above, must be addressed in study subject data collection 
forms.  A completed copy of the DMID Protocol Deviation Form must be maintained in the 
Regulatory File, as well as in the subject's chart.  Protocol deviations must be sent to the local 
IRB/IEC per its guidelines.  The site principal investigator and other study personnel are 
responsible for knowing and adhering to their IRB requirements. 
 

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16 PUBLICATION POLICY 
All investigators funded by the NIH must submit or have submitted for them to the National 
Library of Medicine’s PubMed Central (http://www.ncbi.nlm.nih.gov/pmc/) an electronic version 
of their final, peer-reviewed manuscripts upon acceptance for publication, to be made publicly 
available no later than 12 months after the official date of publication.  The NIH Public Access 
Policy ensures the public has access to the published results of NIH funded research.  It requires 
investigators to submit final peer-reviewed journal manuscripts that arise from NIH funds to the 
digital archive PubMed Central upon acceptance for publication.  Further, the policy stipulates 
that these papers must be accessible to the public on PubMed Central no later than 12 months 
after publication. 
Refer to: 
• 
NIH Public Access Policy, http://publicaccess.nih.gov/ 
• 
NIH OER Grants and Funding, http://grants.nih.gov/grants/oer.htm 
Following completion of the study, the lead principal investigator is expected to publish the 
results of this research in a scientific journal.  The International Committee of Medical Journal 
Editors (ICMJE) member journals have adopted a trials-registration policy as a condition for 
publication.  This policy requires that all clinical trials be registered in a public trials registry such 
as ClinicalTrials.gov* (http://clinicaltrials.gov/), which is sponsored by the National Library of 
Medicine.  Other biomedical journals are considering adopting similar policies. 
The ICMJE defines a clinical trial as any research project that prospectively assigns human 
subjects to intervention or comparison groups to study the cause-and-effect relationship between 
a medical intervention and a health outcome.  Studies designed for other purposes, such as to 
study pharmacokinetics or major toxicity (e.g., Phase I trials), would be exempt from this policy. 
In the event that either the Giardia or E. histolytica portion of the trial is completed earlier than 
the other cohort, data from the completed cohort may be released after the database from that 
portion of the trial is locked. 
It is the responsibility of DMID to register this trial in an acceptable registry.  Any clinical trial 
starting enrollment after 01 July 2005 must be registered on or before subject enrollment.  For 
trials that began enrollment prior to this date, the ICMJE member journals will require registration 
by 13 September 2005, before considering the results of the trial for publication. 
For trials in which DMID is not the IND/IDE sponsor, or there is no IND/IDE, and DMID does 
not provide data management services, it is the responsibility of the investigator to register the 

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trial and post results in compliance with Public Law 110-85, the Food and Drug Administration 
Amendments Act of 2007 (FDAAA). 

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Savioli, Smith H, and Thompson A 2006.Giardia and Cryptosporidium join the ‘Neglected 
Diseases Initiative’. Trends in Parasitol. 22:203-8.  
Solaymani-Mohammed, S. Genkinger, J.M., Loffredo, CA, Singer, S.M. 2010. Meta-analysis of 
the effectives of albendazole compared with metronidazole as treatment of infections with 
Giardia duodenalis. PLoS Neg. Trop. Dis. 4(5): e682.  

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73 
Taniuchi, M., Verweij, J.J., Noor, Z., Sobuz, S.U., van Lieshout, L., Petri, W.A., Haque, R., and 
Houpt. E.R. 2011. High throughput multiplex PCR and probe-based detection with Luminex 
beads for seven intestinal parasites. 2011. Am. J. Trop. Med. Hyg. 84:332-7.  
Tejman-Yarden N, Miyamoto Y, Leitsch D, Santini J, Denath A, Gut J, McKerrow J, Reed SL, 
Eckman L. A reprofiled drug, auranofin, is effective against metronidazole-resistant Giardia 
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Upcroft P, Upcroft JA. 2001. Drug targets and mechanisms of resistance in the anaerobic 
protozoa. ClinMicrobiol. Rev. 14:150-64. 
Wassmann, C., Hellberg, A., Tannich, E., and Bruchhaus, I. 1999. Metronidazole resistance in 
the protozoan parasite Entamoeba histolytica is associated with increased expression of iron-
containing superoxide dismutase and peroxiredoxin and decreased expression of ferredoxin 1 
and flavin reductase. J. Biol. Chem. 274:26051-26056. 
Wright JM, Dunn LA, Upcroft P, Upcroft JA. 2003. Efficacy of antigiardial drugs. Expert Opin 
Drug Saf. 2:529-41.  
Yoder, JS, Harral, C., Beach, MJ. 2010. Giardiasis surveillance- United States, 2006-8, MMWR. 
59:15-25. 
 

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APPENDIX A:   SCHEDULE OF EVENTS 
Procedure 
Visit* 
Number 
00A 
00B 
01 
02 
03 
04 
05 
062 
07 
08 
09 
102 
Study Day  
-4 
-4 
1 
2 
3 
4 
5 
6 
7  
14 
28 
30 
Window 
(days) 
-4 to1 
-4 to 
-1 
+16--- 
--- 
--+1- 
--- 
+16--- 
--- 
+1 
+/-3 
+/-3 
 
+/-3 
Review 
Eligibility 
 
X 
 
 
X 
 
 
 
 
 
 
 
 
 
Informed 
Consent 
 
X 
 
 
 
 
 
 
 
 
 
 
 
Demographic 
Data 
 
X 
 
 
 
 
 
 
 
 
 
 
 
Medical History 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Targeted 
Physical Exam3 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Vital signs4 
 
 
X 
X 
 
 
 
 
 
 
 
 
 
Concomitant 
Medications 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Randomization/
Enrollment 
 
 
 
X 
 
 
 
 
 
 
 
 
 
Assessment for 
side effects of 
treatment 
 
 
 
 
 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
X 
 
AEs/SAEs 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
Urine for 
pregnancy 
testing 
 
 
X 
 
 
 
 
 
 
 
 
 
 
Treatment 
(giardiasis 
subjects) 
 
 
 
X 
X 
X 
X 
X 
 
 
 
 
 

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Treatment (E. 
histolytica)  
 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
 
 
 
Procedure 
 
 
Visit 
Number 
00A 
00B 
01 
02 
03 
04 
05 
063 
07 
08 
09 
104 
Study Day  
-4 
-3 
1 
2 
3 
4 
5 
6 
7  
14 
28 
30 
Window 
(days) 
-4 to 
1 
-3 to 
-1 
+18--- 
--- 
--+18- 
--- 
+18--- 
--- 
+1 
+/-3 
+/-3 
 
+/-3 
Urine Dipstick 
for Proteinuria 
 
 
 
X 
 
 
 
 
 
 
 
X 
 
 
 
Complete 
Blood Count5 
 
 
 
X 
 
 
 
 
 
 
X 
 
 
 
BUN 
 
 
X 
 
 
 
 
 
 
X 
 
 
 
Creatinine 
 
X 
 
 
 
 
 
 
X 
 
 
 
ALT 
 
X 
 
 
 
 
 
 
X 
 
 
 
AST 
 
X 
 
 
 
 
 
 
X 
 
 
 
Stool 
specimen6 
 
X 
 
X 
 
X 
 
X 
 
X 
X 
X 
 
Blood volume 
 
 
17 
 
 
 
 
 
 
17 
 
 
 
Blood volume 
totals 
 
 
17 
 
 
 
 
 
 
34 
 
 
 
Standard of 
Care Treatment 
 
 
 
 
 
 
 
 
 
 
 
 
X 
 
*Subjects will be enrolled at the icddr,b or Rajshahi Hospitals and have follow-up visits until their diarrhea has resolved. Subsequent follow-up visits may be 
held at the icddr,b or Rajshahi Hospital, or local (to Rajshahi) clinic or their home except for Visit 07 which must be performed at icddr,b or Rajshahi Hospital or 
Clinic because blood drawing is required. 
1Visit 06 will only be conducted for subjects receiving treatment for amebiasis. 
2Visit 10 will only be conducted for subjects who remain positive for amebiasis or giardiasis at Day 28 and have not previously received standard of care 
treatment.  
3Targeted physical exam may also include observations for rashes, hair loss, urticaria or glossitis, and an abdominal exam. 
4Vital signs (oral temperature, pulse, blood pressure) 
5Complete blood count includes white blood cells, hemoglobin, and platelet count.  
6Window of + 1 day for stool specimens after (and including) Day 1. Date of collection will be noted. The window does not apply to treatment administration or 
other required assessments. 

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7Amebiasis patients only 

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APPENDIX B:LAB PROCESSING FLOW SHEET: CLINICAL/ 
SAFETY LAB TESTS 
Protocol # 15-0015 
 
Instructions- All specimens must be recorded on a form similar to the one below including the visit time/study ID number, tube type, 
specimen test, processing and storage instructions 
Visit/Time of 
Draw 
Tube Type/Quantity 
Test/Assay/Derivative 
Processing/Aliquot/Storage/Shipping 
Screening 
 
Day -4 to -
1, 
Visit 00A 
 
Stool collection vial for Stool 
Evaluation 
 
Rapid Stool EIA for Giardia 
or E. histolytica 
EIA stool antigen test if 
positive  
Wet mount 
Concentration and 
Trichrome staining 
Qualitative PCR for Giardia 
and Entamoeba 
Stools kept cold and shipped daily to icddr,b or Rajshahi 
hospital- See Lab SOPs 
 
 
Screening  
 
Day -4 to -1, 
Visit 00B 
Urine collection vial 
Dip stick for protein 
Pregnancy testing 
Performed in icddr,b or Rajshahi Hospital or designated 
lab,  
Purple top tube (7mls) 
Complete blood count 
Kept at room temp and transferred same day to lab for 
analysis 
Serum separator tube (10 ml) 
BUN, creatinine, SGOT, 
SGPT 
Kept at room temp and transferred same day to lab for 
analysis 
Left over serum aliquoted and kept at -80oC 
Enrollment 
Day 1,  
Visit 01 
Stool collection vial for Full 
Stool Evaluation (+ 1 Day) 
 
 
 
 
 
 
 
 
 
 
 
 
 
Antigen testing for Giardia 
or E. histolytica 
 
Wet mount 
Concentration and 
Trichrome staining 
 
Qualitative PCR for Giardia 
and Entamoeba 
 
Quantitative PCR for 
Giardia or Entamoeba in 
positive stools 
 
Specimens processed as above 
 
Processed as above 
 
 
Fixed stool stored at room temp  
 
 
Processed as above 
 
Extracted DNA from stool obtained before first 
auranofin dose. Shipped every 1-3months to UCSD for 
quantitative PCR   
Day 3,  
Visit 03  
Stool collection vial for Full 
Stool Evaluation (+1 day) 
Refer to Day 1 (Not 
performed on patients who 
receive metronidazole prior 
to completing dosing 
regimen) 
Refer to Day 1 
Day 5,  
Visit 05 
Stool collection vial for Full 
Stool Evaluation 
(+ 1 day) 
 
Refer to Day 1 (Not 
performed on patients who 
receive metronidazole prior 
Refer to Day 1 
 

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to completing dosing 
regimen) 
Day 7, 
Visit 07 
 
Stool collection vial for Full 
Stool Evaluation (+1 Day) 
 
Refer to Day 1 (Not 
performed on patients who 
receive metronidazole prior 
to completing dosing 
regimen) 
Refer to Day 1 
Urine Collection Vial 
Urine Dip stick for protein 
Refer to Screening Visit 00B 
Purple top tube (7mls) 
Complete blood count and 
differential  
Refer to Screening Visit 00B 
SST (10 ml) 
BUN, Cr, SGOT, SGPT,  
Refer to Screening Visit 00B 
Day 14, 
Visit 08 
+/- 3 days 
Stool collection vial for Full 
Stool Evaluation 
Refer to Day 1 
If positive at Day 14, 
perform parasite genotyping. 
(Not performed on patients 
who receive metronidazole 
prior to completing dosing 
regimen) 
Refer to Day 1 
Stored DNA sent to UVA for parasite genotyping every 
1-3 months 
Day 28, 
Visit 09 
+/- 3 days 
Stool collection vial for Full 
Stool Evaluation 
Refer to Day 1 
If positive at Day 14, 
perform parasite genotyping. 
(Not performed on patients 
who receive metronidazole 
prior to completing dosing 
regimen) 
Refer to Day 1 
Stored DNA sent to UVA for parasite genotyping every 
1-3 months 
 
 
 

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APPENDIX C: ICDDR,B NORMAL LABORATORY VALUES 
(APPLICABLE FOR BOTH SITES) 
 
Male 
Female 
White Blood cells (109/L) 
4.0 –11.0   
same 
Platelets (109/L) 
150.0 –  450.0 
same 
Hemoglobin (gm/dL) 
12.5 –17.5 
11.5 – 16.5  
Blood urea nitrogen (mg/dL) 
5 –24  
same 
Creatinine (umol/L) 
 53-106 
44-97 
AST/SGOT (U/L) 
0.01 – 38.0 
0.01 – 32.0 
ALT/SGPT (U/L) 
0.01 – 41.0 
0.01 – 31.0