Clinical trial NCT03840811
Conditions: Gonococcal Infection
Keywords: ceftriaxone challenge ciprofloxacin males Neisseria gonorrhoeae
Sponsor: National Institute of Allergy and Infectious Diseases (NIAID)
Start date: 2017-04-23; Completion date: 2019-11-25
Enrollment: 27 participants; Allocation: NON_RANDOMIZED; Intervention model: PARALLEL

Brief summary:
This is a Phase 1, interventional, non-randomized, experimental infection model study with healthy adult males adults (N=32) between the ages of 18-36 at study enrollment. The study is designed to test the requirements of predicted N. gonorrhoeae virulence determinants for gonococcal infection in the male urethra. The duration for all participants will be about 3 weeks. Study duration will be approximately about 18 months for implementation and enrollment. The primary objectives of the present study are to: (1) compare infectivity of different isogenic mutants with wild-type (WT) N. gonorrhoeae in noncompetitive infections and to (2) assess relative fitness of the mutant in competitive infections initiated by inocula containing equivalent numbers of both WT and mutant strains for mutants with WT infectivity.

Detailed description:
This is a Phase 1, interventional, non-randomized, experimental infection model study with healthy adult males adults (N=32) between the ages of 18-36 at study enrollment. The study is designed to test the requirements of predicted N. gonorrhoeae virulence determinants for gonococcal infection in the male urethra. For each mutant to be investigated under this protocol, groups of subjects will be enrolled first in noncompetitive infection studies: Group 1 (n = up to 8) will receive a bacterial inoculum containing only the isogenic mutant N. gonorrhoeae strain, and Group 2 (n = up to 8) will receive a bacterial inoculum containing only the wild-type (WT) N. gonorrhoeae strain. If primary and secondary outcomes are not different for the isogenic mutant and WT strains, a group of subjects will be enrolled in competitive infection studies: Group 3 (up to 16) will receive a bacterial inoculum containing a mixture of equivalent numbers the isogenic mutant and WT strain. All subjects will be examined daily for symptoms of infection and receive antibiotic treatment at the end of the inpatient portion of the trial. Within 7 days of antibiotic treatment, subjects will return for a follow-up examination. A final follow-up phone interview will be conducted within 2 weeks of antibiotic treatment. The duration for all participants will be about 3 weeks. Study duration will be approximately about 18 months for implementation and enrollment. The primary objectives of the present study are to: (1) compare infectivity of different isogenic mutants with wild-type (WT) N. gonorrhoeae in noncompetitive infections and to (2) assess relative fitness of the mutant in competitive infections initiated by inocula containing equivalent numbers of both WT and mutant strains for mutants with WT infectivity. Note, competitive infections will not be performed if non-competitive infections are substantially different. If this is the case, there will be no analysis for this primary objective. The study secondary objective is to compare the clinical course of infection with mutant and WT N. gonorrhoeae in noncompetitive infections.

Arms: arm 1: Subjects (n = up to 8) will receive a bacterial inoculum containing only the isogenic mutant N. gonorrhoeae strain arm 2: Subjects (n = up to 8) will receive a bacterial inoculum containing only the wild-type (WT) N. gonorrhoeae strain. arm 3: Subjects (n= up to 16) will receive a bacterial inoculum containing a mixture of equivalent numbers the isogenic mutant and WT strain
Interventions: intervention 1: Cefixime intervention 2: Ceftriaxone intervention 3: Ciprofloxacin intervention 4: Neisseria gonorrhoeae strain FA1090 A26 intervention 5: Neisseria gonorrhoeae strain FA7537
Intervention descriptions: intervention 1: Mandatory rescue therapy consisting of cefixime 400 mg orally in a single dose: on patient request, at the onset of symptoms or on the 5th study day after inoculation. intervention 2: Mandatory rescue therapy consisting of ceftriaxone 250 mg intramuscularly in a single dose: on patient request, at the onset of symptoms or on the 5th study day after inoculation. intervention 3: Mandatory antibiotic treatment failure therapy: Ciprofloxacin 500 mg orally in a single dose: if the subject has a positie test of cure 1 week post initial antibiotic treatment intervention 4: 0.4 mL of a suspension containing 10\^5 - 10\^6 CFU of Neisseria gonorrhoeae, in phosphate-buffered saline, delivered to the anterior urethra through a No.8 pediatric French catheter. intervention 5: 0.4 mL of a suspension containing 10\^5 - 10\^6 CFU of Neisseria gonorrhoeae, in phosphate-buffered saline, delivered to the anterior urethra through a No.8 pediatric French catheter

Protocol documents:
Prot_SAP_000.pdf: 
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Experimental Human Infection with Isogenic Mutants of Neisseria 
gonorrhoeae 
DMID Protocol Number: 09-0106 
Other Identifying Numbers: UNC IRB Study # 12-0482  
DMID Funding Mechanism: NIH grant U01AI114378 
IND Sponsor: National Institute of Allergy and Infectious Diseases 
Principal Investigator: Marcia M. Hobbs, PhD 
Co-Principal Investigator: Joseph A. Duncan, MD, PhD 
DMID Clinical Project Manager: Barbara Hahn, RN, BSN 
DMID Medical Officer: Lori Newman, MD 
DMID Medical Monitor: Mohamed Elsafy, MD 
 
 
 
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STATEMENT OF COMPLIANCE 
The study will be carried out 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 individuals responsible for the design and conduct of this study have completed Human 
Subjects Protection Training. 
 


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SIGNATURE PAGE 
The signature below constitutes the approval of this protocol and the attachments,and provides 
the necessary assurances that this trial will be conducted according to all stipulations of the 
protocol, including all statements regarding confidentiality, and according to local legal and 
regulatory requirements and applicable US federal regulations and ICH guidelines. 
 
Site Investigators: 
 
Signed: 
 
 
 
 
Date: 
 
 
 
Name:   Marcia M. Hobbs, PhD 
Title:      Professor of Medicine 
 
 
 
 
 
 
 
Signed: 
 
 
 
Date: 
 
 
 
Name:   Joseph A. Duncan, MD, PhD 
Title:      Associate Professor of Medicine 
 
 
 
 
 
 


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TABLE OF CONTENTS 
Statement of Compliance 
................................................................................................................ 
ii
Signature Page ............................................................................................................................... iii 
Table of Contents 
........................................................................................................................... iv 
List of Tables 
................................................................................................................................ vii 
List of Abbreviations 
................................................................................................................... viii 
Protocol Summary ...........................................................................................................................x 
1 
Key Roles 
..............................................................................................................................14 
2  Background Information and Scientific Rationale ...............................................................17 
2.1 
Background Information 
...........................................................................................17 
2.2 
Rationale 
...................................................................................................................18 
2.3 
Potential Risks and Benefits 
.....................................................................................19 
Potential Risks ..............................................................................................19
  Known Potential Benefits 
.............................................................................21
3
Objectives .............................................................................................................................22
3.1  Study Objectives 
.......................................................................................................22 
3.2  Study Outcome Measures 
.........................................................................................22
Primary Outcome Measures 
..........................................................................22
Secondary Outcome Measures 
......................................................................23
  Exploratory Outcome Measures ...................................................................23
4  Study Design 
.........................................................................................................................24 
4.1  Substudies 
.................................................................................................................25 
5  Study Enrollment and Withdrawal........................................................................................26 
5.1 
Subject Inclusion Criteria .........................................................................................26
5.2 
Subject Exclusion Criteria 
........................................................................................27
5.3 
Treatment Assignment Procedures 
...........................................................................28
  Randomization Procedures 
...........................................................................28
  Masking Procedures 
......................................................................................28
Reasons for Withdrawal 
................................................................................28
  Handling of Withdrawals 
..............................................................................29
  Termination of Study 
....................................................................................29
6  Study Intervention/Investigational Product 
..........................................................................30 
6.1  Study Product Description 
........................................................................................30 
Acquisition 
....................................................................................................30
  Formulation, Packaging, and Labeling 
.........................................................30
  Product Storage and Stability........................................................................31
6.2  Dosage, Preparation and Administration of Study Intervention/Investigational 
Product 
......................................................................................................................32 


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6.3  
Modification of Study Intervention/Investigational Product for a Participant 
.........33 
6.4  Accountability Procedures for the Study Intervention/Investigational Product(s) 
...33 
6.5  Assessment of Subject Compliance with Study Intervention/Investigational 
Product 
......................................................................................................................34 
6.6 
Concomitant Medications/Treatments 
......................................................................34
7  Study Schedule......................................................................................................................36
7.1 
Screening...................................................................................................................36
7.2 
Enrollment/Baseline 
..................................................................................................37
7.3  Follow-up 
..................................................................................................................37 
7.4 
Final Study Visit 
.......................................................................................................39 
7.5 
Early Termination Visit 
............................................................................................39 
7.6 
Unscheduled Visit 
.....................................................................................................39 
8  Study Procedures/Evaluations ..............................................................................................40 
8.1 
Clinical Evaluations 
..................................................................................................40
8.2  Laboratory Evaluations 
.............................................................................................41 
  Clinical Laboratory Evaluations 
...................................................................41
Special Assays or Procedures 
.......................................................................41
  Specimen Preparation, Handling, and Shipping 
...........................................42
9
Assessment of Safety 
............................................................................................................44
9.1 
Specification of Safety Parameters 
...........................................................................44
9.2  
Methods and Timing for Assessing, Recording, and Analyzing Safety 
Parameters 
.................................................................................................................45
Adverse Events 
.............................................................................................45
Reactogenicity (for Vaccine Studies and Some Therapeutic Trials) 
............46
  Serious Adverse Events 
................................................................................46
  Procedures to be Followed in the Event of Abnormal Laboratory Test 
Values or Abnormal Clinical Findings 
.........................................................49
9.3  Reporting Procedures 
................................................................................................49 
  Serious Adverse Events 
................................................................................49
  Regulatory Reporting for Studies Conducted Under DMID-Sponsored 
IND ...............................................................................................................49
  Regulatory Reporting for Studies Not Conducted Under 
DMID-Sponsored IND..................................................................................50
Other Adverse Events (if applicable) 
............................................................50
  Reporting of Pregnancy 
................................................................................51
9.4  Type and Duration of Follow-up of Subjects after Adverse Events 
.........................51 
9.5 
Halting Rules ............................................................................................................51
9.6  Safety Oversight (ISM plus SMC) 
............................................................................52
10 Clinical Monitoring 
...............................................................................................................53
10.1 Site Monitoring Plan 
.................................................................................................53


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11 Statistical Considerations 
......................................................................................................54
11.1  Study Hypotheses......................................................................................................54 
11.2 Sample Size Considerations 
......................................................................................55
11.3  Planned Interim Analyses (if applicable) 
..................................................................56 
Infectivity Outcomes Review .......................................................................56
Safety Review 
...............................................................................................57
  Immunogenicity or Efficacy Review 
............................................................57
11.4 Final Analysis Plan 
...................................................................................................57
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 
.......................................................................................61
14.3  Informed Consent Process 
........................................................................................61
  Informed Consent/Assent Process (in Case of a Minor) 
...............................62
14.4  Exclusion of Women, Minorities, and Children (Special Populations) 
....................62
14.5 Subject Confidentiality 
.............................................................................................62
14.6  Study Discontinuation 
...............................................................................................63 
14.7  Future Use of Stored Specimens 
...............................................................................63 
15  Data Handling and Record Keeping 
.....................................................................................64 
15.1 Data Management Responsibilities...........................................................................64
15.2 Data Capture Methods 
..............................................................................................64
15.3  Types of Data 
............................................................................................................64 
15.4 Timing/Reports 
.........................................................................................................65
15.5  Study Records Retention...........................................................................................65 
15.6  Protocol Deviations 
...................................................................................................65
16 Publication Policy 
.................................................................................................................66
17 Literature References 
............................................................................................................68
18  Supplements/Appendices 
......................................................................................................70 
18.1 Appendix A: Schedule of Events 
..............................................................................70 
18.2  Appendix B: Subject Comprehension 
.......................................................................72 
18.3  Appendix C: Toxicity Table 
.....................................................................................73


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LIST OF TABLES 
Table 1.  Abnormal vital signs 
...................................................................................................... 38 
Table 2.  Sample size considerations for noncompetitive infection studies 
................................. 55 
 
 


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LIST OF ABBREVIATIONS 
AE 
Adverse Event/Adverse Experience 
ANC 
Absolute Neutrophil Count 
BSE 
Bovine spongiform encephalopathy 
CFAR 
Center for AIDS Research 
CFR 
Code of Federal Regulations 
CFU 
Colony forming unit 
CH50 
Total Complement 
CIOMS 
Council for International Organizations of Medical Sciences 
CONSORT 
Consolidated Standards of Reporting Trials 
CFR 
Code of Federal Regulations 
CRF 
Case Report Form 
CRO 
Contract Research Organization 
CT 
Chlamydia trachomatis, C. trachomatis 
CTRC 
Clinical and Translational Research Center 
DCC 
Data Coordinating Center 
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 
Federal Wide Assurance 
GCB 
Gonococcal culture broth 
GCP 
Good Clinical Practice 
HIPAA 
Health Insurance Portability and Accountability Act 
HIV 
Human Immunodeficiency Virus 
IB 
Investigator’s Brochure 
IBC 
Institutional Biosafety Committee 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
ICMJE 
International Committee
 of Medical Journal Editors 
IDE 
Investigational Device Exemption 
ID
xx 
Infectious dose (xx% of inoculated subjects are expected to 
become infected) 
IEC 
Independent or Institutional Ethics Committee 
IND 
Investigational New Drug Application 
inoc. 
inoculum 
IRB 
Institutional Review Board 
ISM 
Independent Safety Monitor 
JAMA 
Journal of the American Medical Association 
LLN 
Lower limit of normal 


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MedDRA 
Medical Dictionary for Regulatory Activities 
MOP 
Manual of Procedures 
mRNA 
messenger RNA encoding expressed proteins 
mut 
mutant 
N 
Number (typically refers to subjects) 
NAAT 
Nucleic Acid Amplification Test 
NCI 
National Cancer Institute, NIH, DHHS 
NDA 
New Drug Application 
NEJM 
New England Journal of Medicine 
NG 
Neisseria gonorrhoeae, N. gonorrhoeae, the causative agent of 
gonorrhea 
NIAID 
National Institute of Allergy and Infectious Diseases, NIH, 
DHHS 
NIH 
National Institutes of Health 
OCRA 
Office of Clinical Research Affairs, DMID, NIAID, NIH, 
DHHS 
OHRP 
Office for Human Research Protections 
OHSR 
Office for Human Subjects Research 
ORA 
Office of Regulatory Affairs, DMID, NIAID, NIH, DHHS 
PCR 
Polymerase Chain Reaction 
PHI 
Protected Health Information 
PI 
Principal Investigator 
PK 
Pharmacokinetics 
PMN 
Polymorphonuclear cell 
prop’n 
proportion 
QA 
Quality Assurance 
QC 
Quality Control 
RNASeq 
Sequence data derived from human or bacterial mRNA 
RT PCR 
Reverse transcriptase PCR 
SAE 
Serious Adverse Event/Serious Adverse Experience 
SMC 
Safety Monitoring Committee 
SOP 
Standard Operating Procedure 
STI 
Sexually Transmitted Infection 
TV 
Trichomonas vaginalis, T. vaginalis 
UNC 
University of North Carolina at Chapel Hill 
US 
United States 
ULN 
Upper limit of normal 
VIM 
Virology, Immunology & Microbiology Core Laboratory 
WBC 
White blood cell 
WHO 
World Health Organization 
WNL 
Within normal limits 
WT 
Wild type 
 


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PROTOCOL SUMMARY 
Title: 
Experimental Human Infection with Isogenic Mutants of Neisseria 
gonorrhoeae 
Phase: 
Phase I 
Population: 
Up to 32 male subjects, > 18 and < 36 years old, living in central 
North Carolina, in general good health with no history of STIs.   
Number of Sites: 
Single site: University of North Carolina at Chapel Hill 
Study Duration: 
18 months 
Subject Participation 
Duration: 
3 weeks including 6 days in the CTRC, a follow-up visit 1 week 
after discharge from the CTRC and a follow-up telephone call 2 
weeks after discharge from the CTRC. 
Description of Agent or 
Intervention: 
Subjects will receive up to 0.4 mL of a suspension containing 105 - 
106 CFU of Neisseria gonorrhoeae, in phosphate-buffered saline, 
delivered to the anterior urethra through a No.8 pediatric French 
catheter.   
Objectives: 
 
Primary: 
 
• Compare infectivity of different isogenic mutants with 
wild-type (WT) N. gonorrhoeae in noncompetitive 
infections.   
• For mutants with WT infectivity, assess relative fitness of 
the mutant in competitive infections initiated by inocula 
containing equivalent numbers of both WT and mutant 
strains.  Note, competitive infections will not be performed 
if non-competitive infections are substantially different.  If 
this is the case, there will be no analysis for this primary 
objective.    
Secondary: 
• Compare the clinical course of infection with mutant and 
WT N. gonorrhoeae in noncompetitive infections.   
 
Exploratory: 


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• Characterize host immune responses during experimental 
gonococcal infection.   
• Characterize bacterial gene expression during experimental 
gonococcal infection.   
 
Description of Study 
Design: 
This is an experimental infection model study designed to test the 
requirements of predicted N. gonorrhoeae virulence determinants 
for gonococcal infection in the male urethra. Subjects will receive 
a suspension containing 10
5 – 10
6 CFU of  N. gonorrhoeae 
delivered to the anterior urethra through a small diameter catheter.  
Inoculation takes place in the inpatient unit of the UNC Clinical 
Translational Research Center (CTRC).  During the inpatient 
portion of the trial, subjects may leave the clinical research ward 
provided they sign out, identify their whereabouts when not on the 
ward, carry a cell phone, and return by 9pm each evening to stay 
overnight on the ward. All subjects will be examined daily for 
signs or symptoms of infection, provide daily first-void urine 
specimens for gonococcal culture to monitor infection and receive 
100% effective antibiotic treatment (cefixime or ceftriaxone) at the 
end of the inpatient portion of the trial, whether infected or not.  
Within 7 days of antibiotic treatment, all subjects return for a test 
of cure and follow up examination. A final follow-up interview is 
conducted with all subjects by phone within 2 weeks of antibiotic 
treatment.   
 
For each mutant to be investigated under this protocol, groups of 
subjects will be enrolled first in noncompetitive infection studies: 
Group 1  (n = up to 8 evaluable subjects) will receive a bacterial 
inoculum containing only the isogenic mutant N. gonorrhoeae 
strain, and Group 2 (n = up to 8 evaluable subjects) will receive a 
bacterial inoculum containing only the WT N. gonorrhoeae strain.  
If primary and secondary outcomes are not different for the 
isogenic mutant and WT strains, a group of subjects will be 
enrolled in competitive infection studies:  Group 3 (up to 16 
evaluable subjects) will receive a bacterial inoculum containing a 
mixture of equivalent numbers the isogenic mutant and WT strain.  
A competitive advantage for one strain during urethral infection 
will be manifest by recovery of that strain in a statistically 
significantly higher proportion of isolates recovered from infected 
subjects than in the inoculum.   
 


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This protocol describes (1) the overall experimental infection 
program with fixed details that do not change with the specific 
gonococcal strains to be studied, including study recruitment, 
screening, enrollment and experimental infection and monitoring 
procedures, and (2) the MtrD study including specific details of the 
experimental inocula and outcome measures to test the 
requirement for the MtrD component of the gonococcal Mtr efflux 
pump during experimental urethral infection.  For all future studies 
designed to test other N. gonorrhoeae strains, a protocol 
amendment will be submitted including the rationale, details of the 
experimental inoculum, outcome measures and consent forms 
specific for each study. The study population of up to 32 subjects 
reflects the sample size required for the MtrD study and includes 
the total number of subjects to be enrolled in noncompetitive and 
competitive infection groups for this mutant.  
Estimated Time to 
Complete Enrollment: 
18 months 
 
 
 


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Schematic of Study Design: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Note, the total number of subjects in Groups 1, 2 and 3 combined will not exceed 32.  Outcomes 
for Groups 1 and 2 will determine whether Group 3 will be enrolled for each isogenic mutant.


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1 KEY ROLES 
 
Individuals: 
Clinical Project Manager:   
Barbara Hahn, RN, BSN 
Nurse Consultant/Clinical Project Manager 
Sexually Transmitted Infections Branch 
DMID, NIAID, NIH  
5601 Fishers Lane, Room E49 
Bethesda, MD 20892-9825 
(if using FedEx or UPS, use zip code 20891) 
Tel: (240) 627-3267 
Email: bhahn@niaid.nih.gov  
 
 
Medical Officer: 
Lori M. Newman, MD 
DMID/NIAID/NIH 
5601 Fishers Lane, Room 8E45, Rockville, MD 20852 
Phone: (301) 761-6958 
Email: lori.newman@nih.gov 
 
Principal Investigator:   
Marcia M. Hobbs, PhD 
Associate Professor of Medicine, Microbiology & Immunology 
University of North Carolina at Chapel Hill 
Infectious Diseases Research 
111 Mason Farm Road, 8309 MBRB CB# 7031 
Chapel Hill, NC 27599 
Phone: (919) 843-6893 
Fax: (919) 843-1015 
Email: mmhobbs@med.unc.edu 
 
Co-Principal Investigator: 
Joseph A. Duncan, MD, PhD 
Associate Professor of Medicine and Pharmacology 
University of North Carolina at Chapel Hill 
Infectious Diseases Research 
111 Mason Farm Road, 8335 MBRB CB# 7031 
Chapel Hill, NC 27599 
Phone: (919) 843-0715 
Fax: (919) 843-1015 
Email: joseph_duncan@med.unc.edu 
 


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Medical Monitor: 
Mohamed M. Elsafy, MD 
Medical Officer 
NIH/NIAID/DMID/OCRA 
5601 Fishers Lane, Room 7E56 
Bethesda, MD 20892-9825 
Phone: (240) 292-4241  
Email: mohamed.elsafy@nih.gov 
 
Institutions: 
Clinical site of volunteer screening, and follow-up visits: 
NC TraCS Institute – CTRC Outpatient Unit 
University of North Carolina at Chapel Hill 
160 Dental Circle 
Burnett-Womack Bldg, 1st Floor 
Room 1036, CB# 7064 
Chapel Hill, NC 27599-7064 
Contact: Janette Goins, RN, BSN, Director of Nursing 
Phone: (919) 843-1070 
Fax: (919) 966-1576 
Email: janette_goins@med.unc.edu 
 
Clinical site of subject enrollment, experimental procedures 
and follow-up visits: 
NC TraCS Institute – CTRC Inpatient Unit 
University of North Carolina at Chapel Hill  
101 Manning Drive, CB# 7600 
Chapel Hill, NC 27599-7600 
Contact: Janette Goins, RN, BSN, Director of Nursing 
Phone: (919) 843-1070 
Fax: (919) 966-1576 
Email: janette_goins@med.unc.edu 
 
Clinical Laboratory: 
McLendon Clinical Laboratories 
University of North Carolina Hospitals 
Chapel Hill, NC 27514 
Contact: Herbert C. Whinna, MD, PhD, Director 
Phone: (919) 966-2318 
Fax: (919) 966-6407 
Email: 
herb_whinna@med.unc.edu 


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Optional: 
 
Co-Investigators intervening with subjects: 
Susan Blevins, ANP-C  
Joseph Eron, Jr., MD; David Margolis, MD; David Wohl, MD; 
Michelle Floris-Moore, MD, MS; Cynthia Gay, MD, MPH;  
Christopher Hurt, MD; Claire Farel, MD 
 
Co-Investigators not intervening with subjects: 
William M. Shafer, PhD (Emory University) 
 
UNC Institutional Review Board: 
CB 7097 
720 Martin Luther King Jr. Blvd. 
Bldg # 385, Second Floor 
Chapel Hill, NC 27599-7097 
Contact: Cassandra Myers, Director 
Phone: (919) 966-3113 
Fax: (919) 966-7879 
Email: irb_questions@unc.edu 
 
Independent Safety Monitor: 
David J. Weber, MD 
130 Mason Farm Road, CB#7030 
2153 Bioinformatics 
Chapel Hill, NC 27599 
Phone: (919) 966-2536 
Fax: (919) 966-1451 
pager: (919) 347-0639 
Email: dweber@unch.unc.edu  
 
Statistical Data Coordinating Center (SDCC) 
The Emmes Corporation 
SDCC Project Manager: Amy Price, MS 
SDCC Data Manager: Logan Richlak 
SDCC Statistician: Amy Tan, MS  
401 N. Washington St., Suite 700 
Rockville, MD 20850 
Phone: (301) 251-1161 
Email: sti@emmes.com 
 
 


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2 BACKGROUND INFORMATION AND SCIENTIFIC 
RATIONALE 
2.1 Background Information   
Gonorrhea remains a major global health concern. With over 106 million cases estimated by 
WHO in 2008, world-wide incidence of gonorrhea surpassed that of chlamydia as the most 
common bacterial sexually transmitted infection (STI).(1) The CDC reported over 350,000 cases 
of Neisseria gonorrhoeae infection in the US in 2014, a 5% increase compared to 2013.(2) 
 
Uncomplicated gonorrhea can be asymptomatic or manifest as urethritis in men and cervicitis in 
women. Complications of untreated gonococcal infections include orchitis, epididymitis and 
pelvic inflammatory disease. Furthermore, like other inflammatory mucosal infections, 
gonorrhea also facilitates HIV transmission and acquisition.(3-5) Appropriate treatment with 
effective antimicrobials is essential for control of gonorrhea, and N. gonorrhoeae (the 
gonococcus or GC) has a long history of emerging antimicrobial resistance, reducing the 
effectiveness of antibiotics used to treat infection. In 2012, the CDC responded to recent 
dramatic decreases in susceptibility to cefixime among circulating GC strains by recommending 
against the use of oral cephalosporins as first-line treatment for gonorrhea.(6) Currently, 
increasing GC resistance to the extended-spectrum injectable cephalosporin, ceftriaxone, 
threatens the last remaining first-line treatment option.(7) With few new antimicrobial therapies 
in the pipeline and no effective GC vaccine, fears of incurable gonorrhea are all too real. The 
potential adverse reproductive health consequences of untreatable GC and attendant increases in 
HIV transmission in areas where both infections are prevalent is alarming.(8)  New therapeutic 
and vaccine targets for gonorrhea are urgently needed.     
Limited in nature to infection of humans, N. gonorrhoeae are equipped with multiple 
mechanisms to subvert our immune responses, and natural GC infection does not result in 
protective immunity. We hypothesize that mutations that alter or eliminate GC mediators of 
resistance to human innate immune responses will reduce infectivity, decrease inflammation or 
increase clearance compared to wild-type (WT) GC. In this clinical trial, we will test the 
requirement for gonococcal structures that mediate resistance to innate host.   
The MtrC-MtrD-MtrE efflux pump endows gonococci with a mechanism to export hydrophobic 
antibiotics(9) and host-derived antimicrobials (antimicrobial peptides, bile salts and 
progesterone) that bathe mucosal surfaces.(10, 11)  The proteins that form the pump are encoded 
by the mtrCDE operon, which is transcriptionally regulated by cis- and trans-acting regulatory 
control processes.(12, 13) Loss-of-function mutations in mtrCDE significantly reduce the ability 
of gonococci to infect the lower genital tract of female mice.
14 Moreover, a null mutation in the 
mtrR gene, which encodes a transcriptional repressor of mtrCDE, or mutations in the DNA 
sequence upstream of mtrCDE enhance both antimicrobial resistance
13 and in vivo fitness in the 
mouse infection model.(14)First with an isogenic mtrD mutant and then with an isogenic mtrE 
mutant, we will examine the role of the mtr system in the experimental human infection model. 
Only the mtrD mutant will be tested under this version of the protocol; modifications will be 
submitted as an amendment for future mutants to be tested.  


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A major limitation in the development of gonococcal vaccines has been the lack of an animal 
model.  We and others have developed a model of experimental human gonococcal infection 
(reviewed by Hobbs et al.(15)) The model takes advantage of the self-limited nature of 
gonococcal urethral infection in male subjects.  Asymptomatic infection can be treated very 
efficiently and quickly.  Urethral infection can be initiated in male volunteer subjects through 
urethral inoculation, and the experimental infection model has proven to be a safe and efficient 
means of studying gonococcal infection in men.(15) To date, over 200 subjects have been 
studied in experimental gonococcal infection trials at UNC without serious complications.(15)  
 
The experimental infection model has been used in several different ways to study the roles of 
putative N. gonorrhoeae virulence determinants in male urethral infection.  Early observational 
studies with wild-type strain FA1090 established infectious doses for this well-described 
strain(15) and characterized antigenic variation of outer membrane proteins during infection.(16, 
17) In later studies to compare the infectivity of WT gonococci with that of isogenic mutants 
lacking gonococcal virulence factors, individual subjects were inoculated with similar doses of 
wild-type or mutant bacteria in noncompetitive infection studies.  These studies demonstrated 
that substantial differences in infectivity between strains could be detected with statistical 
significance using this model with reasonable numbers of subjects.  Competitive infection with 
mixed inocula increases the sensitivity of the model allowing detection of more subtle 
differences in gonococcal virulence than can be demonstrated in noncompetitive infections with 
a single test strain.(18, 19)   
2.2 Rationale 
Despite the robust inflammation that characterizes symptomatic gonorrhea, natural GC infection 
does not confer protection from subsequent infection.  Limited to human hosts in nature, GC are 
highly adapted to evade our innate defenses.  Results of experimental infection studies in the 
female mouse model have informed the selection of isogenic mutants to be tested in this clinical 
trial.  However, the natural restriction of N. gonorrhoeae infection to human hosts limits the 
extent to which animal models can predict results that are clinically translatable.  The 
experimental human infection model is essential for testing hypotheses regarding the roles of 
putative virulence factors.  The rationale for this study is that the human challenge model so 
closely resembles natural infection that the results will provide a compelling agenda for scientific 
research and greatly facilitate vaccine development.  We hypothesize that key virulence 
determinants involved in N. gonorrhoeae resistance to innate immunity are essential for 
infection in the male urethra.  We predict that mutations abolishing expression of these 
virulence determinants will eliminate or significantly reduce gonococcal infectivity, thus 
identifying potential vaccine candidates. 
 


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2.3 Potential Risks and Benefits 
Potential Risks  
Subjects will provide blood samples via venipuncture. Risks of venipuncture include:  
 acute pain and discomfort  
 development of a bruise 
 formation of a blood clot in the vein that has been stuck 
 infection   
 Rarely, blood donors become faint while blood is being obtained.   
Subjects may experience some discomfort associated with the urethral catheterization during 
inoculation including mild irritation or itching for several minutes after the procedure.  Risks of 
gonococcal urethritis include urethral inflammation and the possibility of epididymitis, orchitis, 
gonococcal bacteremia with attendant meningitis and endocarditis, and immunological responses 
to infection.  The risks of serious complications from urethral gonococcal infection are extremely 
rare, even in natural infection.  Careful screening and observation during participation, habitation 
in the Clinical and Translational Research Center (CTRC), immediate treatment of signs or 
symptoms of urethritis and treatment prior to discharge from the CTRC are all designed to 
minimize the possibility of complications.  Among the 219 subjects previously studied, one 
subject was discovered to have asymptomatic hematuria and one subject experienced dysuria 
relieved with pyridium.  No other complications have been noted. 
The research laboratory follow rigorous quality control procedures, nevertheless, the plates on 
which gonococcal inocula are grown may become contaminated with other bacteria.  In 58 
different experimental trials including 219 individual subjects, contaminants were found in the 
inoculum once (1.7% of the time).  In one trial, five subjects received contaminated bacterial 
suspensions. One day after inoculation, we realized that the subjects had been inoculated with 
contaminants, identified as Staphylococcus species.  The trial was stopped, and subjects were 
treated with ceftriaxone for possible gonorrhea. To treat possible staphylococcal infection, four 
subjects received a 3-day course of Trimethoprim/Sulfamethoxazole (Septra).  The fifth subject 
reported a sulfa allergy and was treated with a 3-day course of Nitrofurantoin (Macrodantin).  
Subjects returned for follow-up examinations one week after the trial and presented without 
complaints, signs or symptoms of urethritis; urinalysis results for all subjects were normal.  
In response to the adverse event described above, protocol procedures were carefully reviewed 
and the following corrective and preventive measures have been instituted to reduce the 
likelihood of similar contamination in future trials.  First, all agar plates containing gonococcal 
growth for use in human challenge experiments shall be thoroughly examined using a dissecting 
microscope; plates will be examined for at least one full minute each.  Plates containing any 
contaminating colonies will not be used to prepare the inoculum suspension.  Second, in addition 
to quantitative cultures of the inoculum suspension plated on GCB agar to enumerate the dose of 
gonococci delivered, we will plate the inoculum suspension, undiluted, on LB agar, which does 
not support the growth of N. gonorrhoeae, but does support the growth of common bacterial 
contaminants (including Staphylococcus and Streptococcus species).  In the event that 


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contaminating bacteria are detected by growth on LB agar, urine specimens will be obtained 
from subjects as soon as they return to the research unit for the first overnight stay.  Specimens 
will be cultured and growth identified to guide appropriate antibiotic treatment.  As soon as this 
information is available, subjects will be treated appropriately for the contaminating bacteria and 
for potential gonococcal infection.  
For experimental gonococcal infections, subjects are treated with a third-generation 
cephalosporin: cefixime (single 400 mg oral dose) or ceftriaxone (single 250 mg dose delivered 
intramuscularly) when infection is clinically apparent, defined by reported symptoms of 
urethritis including urethral discharge or dysuria, plus the recovery of viable N. gonorrhoeae 
from urine or urethral swab cultures, or at the end of the inpatient portion of the trial, whichever 
comes first.  In the unlikely event of a treatment failure, alternative treatment would be with a 
fluoroquinolone (ciprofloxacin, single 500 mg oral dose).   Although monotherapy with oral 
cephalosporins or fluoroquinolones including ciprofloxacin is no longer recommended by the 
CDC for treatment of naturally-acquired gonorrhea, all N. gonorrhoeae strains included in this 
protocol have documented susceptibility to all antibiotics listed in the protocol that could 
potentially be used to treat experimental gonococcal infection.  Coagulopathy and bleeding have 
been reported with third-generation cephalosporins, including cefixime and ceftriaxone.  
Potential side effects including nausea, diarrhea, anorexia can occur, particularly in children.  
Hematologic reactions are rare, and none of the third-generation cephalosporins is significantly 
nephrotoxic.(20)  Although an increased risk of allergic reaction among patients with penicillin 
allergy has been observed for some cephalosporins, this risk is quite low for third-generation 
cephalosporins including cefixime and ceftriaxone.(21)  The most frequent reactions to 
cephalosporins are non-pruritic, non-urticarial rashes, which occur in less than 3% of patients.   
Peripheral neurophathy is an identified risk of fluoroqinolones, including ciprofloxacin; rapid 
onset and permanent damage are possible.  In addition, fluoroquinolones are rarely associated 
with tendinopathy, such as Achilles tendon rupture.(22)  Allergic reactions, including serious 
anaphylactic reactions, and central nervous system events, including convulsions, confusion, 
tremors, hallucinations, and depression, have been reported rarely in patients receiving 
fluoroquinolone antibiotics including ciprofloxacin. Common side effects of ciprofloxacin, 
which are usually mild, include nausea, headache, dyspepsia, dizziness, vaginal yeast infection 
and diarrhea.  It is also possible for fluoroquinolones to cause vomiting, rash, and abdominal 
discomfort/pain.  Some patients taking fluoroquinolones may become more sensitive to sunlight 
or ultraviolet light.  On 12 May 2016, the FDA advised that the serious side effects associated 
with fluoroquinolone antibacterial drugs generally outweigh the benefits for patients with 
uncomplicated urinary tract infections who have other treatment options.  However, if needed in 
case of cephalosporin treatment failure, a single dose of ciprofloxacin would be used, which 
minimizes risks other than allergic reactions. 
Transmission of N. gonorrhoeae to another individual is also a risk of the study, but this has not 
occurred in previous experimental infection studies at UNC in which 120 subjects were 
determined to be infected.  Subjects sleep in the inpatient unit of the CTRC, and signed consent 
forms specifically instruct participants to abstain from all sexual activity until the completion of 
the study and the follow-up test for gonorrhea is negative.   


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Known Potential Benefits 
Direct benefits to subjects are limited to the possible discovery of a previously unrecognized 
medical condition.  In addition, the information generated from the study will allow us to 
examine hypotheses that individual gonococcal gene products play a role in the ability of N. 
gonorrhoeae to establish infection in the human host.  These studies will establish whether 
specific protein expression is required for urethral infection and may provide a rationale for a 
gonococcal vaccine. 


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3 OBJECTIVES 
3.1 Study Objectives 
Primary:
 Compare infectivity of different isogenic mutants with wild-type (WT) N. gonorrhoeae in 
noncompetitive infections.   
 For mutants with WT infectivity, assess relative fitness of the mutant in competitive 
infections initiated by inocula containing equivalent numbers of both WT and mutant 
strains.  Note, competitive infections will not be performed if non-competitive infections 
are substantially different.  If this is the case, there will be no analysis for this primary 
objective.    
Secondary: 
 Compare the clinical course of infection with mutant and WT N. gonorrhoeae in 
noncompetitive infections.   
Exploratory: 
 Characterize host immune responses during experimental gonococcal infection.   
 Characterize bacterial gene expression during experimental gonococcal infection.   
 
3.2 Study Outcome Measures 
Primary Outcome Measures 
To compare the ability of mutant gonococci to infect the male urethra, we will first conduct 
noncompetitive infection studies with inocula containing mutant or wild-type N. gonorrhoeae.  
For infections initiated with individual N. gonorrhoeae strains, the primary outcome measure is: 
 The proportion of subjects in each group that become infected by day 6 as defined by a 
positive urine or urethral swab culture.   
For isogenic mutants that do not demonstrate significantly lower infectivity compared to WT N. 
gonorrhoeae, we will assess relative fitness of the mutant in competitive infections initiated by 
inocula containing equivalent numbers of both WT and mutant strains.  Competitive infections 
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using the smallest possible number of subjects.  For infections initiated with mixed inocula, the 
outcome measures are:  
 The proportion of subjects that become infected by day 6 as defined by a positive urine 
or urethral swab culture.  
 The competitive index (CI) of the mutant compared to WT proportion of organisms with 
the predicted competitive advantage recovered from urine and/or urethral swab 
specimens from among individual infected subjects, defined by the ratio of colony 
forming units (cfu) of the two strains recovered from urine cultures on the day of 
treatment (output) compared to the ratio of strains in the inoculum (input). CI = mutant 
cfu(output)/wild-type cfu(output) ÷ mutant cfu(input)/wild-type cfu(input).  
Secondary Outcome Measures
To compare the clinical course of infection with mutant and WT N. gonorrhoeae in non-
competitive infections, the outcome measures are: 
 The occurrence of signs and symptoms of urethritis attributable to gonococcal infection 
by day 6  
 The occurrence of bacteriuria (Log10 cfu N. gonorrhoeae/mL urine sediment) on day of 
treatment 
 The occurrence of urethritis (> 5.8 Log10 WBC/mL urine sediment) on day of treatment 
 Median time from inoculation to treatment among infected subjects by day 6, by strain 
Exploratory Outcome Measures 
To characterize host immune responses during experimental gonococcal infection, the outcome 
measures are: 
 The detection of local cytokines in urine and systemic cytokines in blood 
 Expression of human genes identified by RNASeq in urine sediment and whole blood 
To characterize bacterial gene expression during experimental gonococcal infection, the outcome 
measure is:  
 Expression levels of specific bacterial genes in urine sediment 


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4 STUDY DESIGN 
Subject Recruitment.   
Healthy adult men, > 18 and < 36 years old, will be recruited into the study by flyer, newspaper, 
radio or electronic advertisements for participation in a bacterial infection study.  Potential 
subjects are given preliminary information about the study by phone.  Interested subjects then 
undergo a screening examination and lab tests to provide further information.  Written informed 
consent is obtained both at the screening visit and upon admission to the Clinical and 
Translational Research Center (CTRC) before inoculation, following thorough discussion with 
study investigators.  Recruitment, screening, and follow-up visits will be conducted in the 
outpatient unit of the CTRC; trial procedures will be conducted in the inpatient unit, with 
provisions for subjects to leave the unit during the day.  Inclusion and exclusion criteria are 
described in detail in sections 5.1 and 5.2. 
 
Experimental procedures. 
 
During the screening visit, the consent form is reviewed in detail, and all aspects of the study are 
discussed.  The subject is given ample time to address questions and concerns.  If the subject 
agrees to participate, written consent is obtained for the main study and subjects initial a 
statement to indicate permission for specimen storage, and a medical history and complete 
physical are performed.  Up to 30 mL of blood and a urine sample are obtained.  Screening lab 
work includes: a complete blood count (CBC) with differential (for WBC count, ANC, and 
hemoglobin level), serum ALT, serum creatinine, HIV, syphilis, HBV and HCV serologies, 
CH50 test for complement deficiency, urinalysis and urine nucleic acid amplification testing for 
chlamydia, gonorrhea and trichomoniasis.  Screening precludes enrollment of subjects with 
health problems, urethral inflammation, STIs, and factors such as complement deficiency that 
might lead to complications of a local infection.  If the results of CBC, serum ALT, creatinine or 
urinalysis obtained at initial screening are outside acceptable limits, and the clinician judges the 
deviation unlikely to be clinically relevant, one time repeat screening is permitted. If the results 
of initial or repeat screening tests are within acceptable limits, the subject may be enrolled in the 
next scheduled trial within 30 days of screening.  Within one week prior to the inoculation visit, 
subjects are sent a confirmation letter or email with directions to return to the outpatient unit of 
the CTRC to provide a urine specimen for repeat STI testing and blood for determination of 
baseline host immune responses 2-4 days before the start date and for presentation at UNC 
Hospital on the start date. 
   
For inoculation, subjects recline on a hospital bed.  The pelvic area is covered with a waterproof 
plastic drape, and the penis is extended through a hole in the drape.  The physician dons sterile 
gloves immediately before the procedure, as does the subject.  The skin around the urethra is 
cleansed with 70% isopropanol and allowed to dry.  Bacterial suspensions in phosphate-buffered 
saline will be delivered to the anterior urethra through a No.8 pediatric French catheter, inserted 
5 cm into the urethra.  The PI or designee will load the inoculum into a dedicated pipette, and a 
licensed physician wearing sterile gloves, will moisten the catheter using sterile saline, insert the 
catheter and deliver the inoculum using aseptic technique.  Inoculation takes only a few seconds 


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and produces minimal discomfort.  Subjects are instructed to apply gentle pressure to the end of 
the penis for 30 minutes to minimize leakage of the inoculum.   
 
Each day after inoculation, subjects are examined between 6:30am and 7:30am for signs or 
symptoms of urethritis, and a first-void urine specimen of 50 mL is collected for gonococcal 
culture, urine dipstick analysis, and microscopic examination of urine sediment for PMNs as an 
indicator of urethral inflammation.  On the day on which a subject is treated, a urethral swab will 
be collected from each subject for gonococcal culture.  All subjects receive antibiotic treatment 
before discharge from the study.  Subjects are treated during the inpatient portion of the study 
when (1) requested by an individual subject, regardless of signs, symptoms or positive cultures 
(2) purulent urethral discharge is observed by the examining clinician or reported by the subject 
or (3) prior to discharge from the CTRC, whether infected or not.  Antibiotics are administered 
by a CTRC nurse in an inpatient room of the research unit; subjects will be observed for 
approximately 20 minutes following administration of study antibiotics. Subjects return to the 
CTRC in one week for reexamination and urine testing by nucleic acid amplification and for 
compensation. 
 
Results from urine cultures are available 24-48h after specimens are plated, however, a positive 
urine culture in the absence of signs or symptoms does not necessitate immediate treatment.  
Among previous experimentally infected subjects, 68% (71/104) had positive urine cultures on 
day 2.  A minority of infected subjects (8/107, 7.5%) reported symptoms (ranging from a slight 
tingling sensation to dysuria) on day 2.  Most positive urine cultures (239/269, 89%) were from 
days without reported symptoms.  Bacterial counts from positive cultures generally increased 
from day 2-6, and the proportion of subjects with positive cultures who were symptomatic 
increased from 6% to 22% on days 2-5 (2 P = 0.035).  However, by day 6, 95% of remaining 
infected subjects were asymptomatic. 
 
4.1 Substudies 
None 
 
 


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5 STUDY ENROLLMENT AND WITHDRAWAL 
The study population includes healthy men > 18 and < 36 years old.  Due to potential 
complications from ascendant gonococcal infection, women cannot safely be included.  Children 
under the age of 18 are inappropriate for inclusion in a study of a sexually transmitted infection.  
The ethnic and racial composition of the study population is expected to reflect those features of 
the UNC undergraduate, graduate and medical student populations, from which most subjects 
will be recruited.   
 
For the MtrD Study, we will enroll up to 32 subjects.  Potential male subjects will be recruited 
from the UNC undergraduate, graduate and medical student populations and from neighboring 
communities by flyer, newspaper and/or electronic advertisements for participation in a bacterial 
infection study.   
 
5.1 Subject Inclusion Criteria 
Subjects must meet all the inclusion criteria to participate in the study. 
 
1. Healthy man > 18 and < 36 years old 
2. Able and willing to be located easily by providing street address and telephone 
number (land line and/or cell phone number)  
3. Willingness to provide written informed consent 
4. Able and willing to attend all study visits including 6-day stay in the CTRC 
during the trial (with ability to leave the unit during the day) and follow-up visit 
during the week after treatment 
5. Able and willing to abstain from all sexual activity until completion of the study 
and the follow-up test for gonorrhea is negative 
6. Acceptable medical history by screening evaluation 
7. No clinically significant abnormalities on physical exam 
8. Urinalysis: leukocyte esterase and WBC values within normal limits 
9. CH50 WNL  
10. Urine negative for chlamydia, gonorrhea, and trichomonas  
11. Negative HIV, syphilis, and HCV test results 
12. Negative HBV core and surface antibodies or results consistent with 
immunization (negative HBV core antibody/positive HBV surface antibody)  
13. Denies history of STIs including gonorrhea, chlamydia, syphilis, HIV, HBV, 
and HCV 
14. Denies history of bleeding diathesis 
15. Denies history of seizures (due to reports of seizures with ciprofloxacin) 
16. Denies history of cancer, except basal cell carcinoma of the skin >5 years ago 
17. Denies history of drug abuse  
18. Denies history of genitourinary surgery 


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5.2 Subject Exclusion Criteria 
1. Subjects meeting any of the exclusion criteria at screening will be excluded 
from study participation. Laboratory values are based on the Toxicity Table in 
Appendix C.Student or employee under the direct supervision of any of the 
study investigators 
2. Any known immunodeficiencies including complement deficiency, antibody 
deficiency, chronic granulomatous disease or HIV infection 
3. Psychiatric disorders that, in the opinion of the physician, would interfere with 
the integrity of the data or volunteer safety  
4. Unstable depression (defined as receiving either <3 months of the same 
medication (and dose) or a decompensating event during the previous 3 months) 
or depression that, in the opinion of the investigator, will compromise the 
subject’s ability to comply with protocol requirements 
5. Heart murmur or heart disease 
6. Anatomic abnormality of the urinary tract 
7. Any antibiotic treatment in the past 30 days, or azithromycin in the past 60 days 
8. Self-reported chemotherapy within the past year 
9. Current steroid use, except for topical application 
10. Allergy to penicillin, cephalosporins or ciprofloxacin or to lidocaine 
11. Treatment with medications in the previous month that are contraindicated with 
cefixime, ceftriaxone or ciprofloxacin and that cannot be withheld for the single 
doses given in this study 
12. Serum creatinine level < 0.7 or > 1.75 mg/dL and deemed clinically significant 
by the study physician 
13. Serum ALT level < LLN or > 105 U/L and deemed clinically significant by the 
study physician 
14. WBC count < 2.5 or > 15.0 x109/L and deemed clinically significant by the 
study physician  
15. Absolute neutrophil count (ANC) < 1.5 or > 7.5 x 109/L and deemed clinically 
significant by the study physician 
Exception: For African Americans, ANC values as low as 1.3 x 10
9/L will be 
allowed (25) 
16. Hemoglobin level < 12.0 g/dL or above ULN and deemed clinically significant 
by the study physician 
17. Urinalysis: Qualitative protein level > 1+ or RBC count > 10/hpf 
 
Medications not permitted with cefixime or ceftriaxone: 
• Warfarin 
• Probenecid 
• Aspirin 
• Diuretics such as furosemide 
• Aminoglycoside antibiotics 
• Chloramphenicol 
 
Medications not permitted with ciprofloxacin: 


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 Tizanidine 
 Theophylline 
 Warfarin 
 Glyburide 
 Cyclosporine 
 Probenecid
 Phenytoin 
 Methotrexate 
 Antacids, multivitamins, and other dietary supplements containing magnesium, 
calcium, aluminum, iron or zinc 
 Caffeine-containing medications 
 Sucralfate or didanosine chewable or buffered tablets 
 
5.3 Treatment Assignment Procedures 
Randomization Procedures 
This is not a randomized trial.  In cohorts (up to 8 subjects enrolled on the same date) for 
noncompetitive infection groups (up to 8 subjects per group as shown in Schematic of Study 
Design on p. xii), all subjects enrolled on a given date will receive the same inoculum suspension 
containing either mutant or WT N. gonorrhoeae.  In cohorts for competitive infection groups (up 
to 16 subjects as shown in Schematic of Study Design on p. xii), all subjects will receive the 
same inoculum suspension containing approximately equal numbers of the two bacterial strains 
under investigation.  The identity of the suspension to be used for each cohort is determined by 
the results of previous studies.  Subjects will not know which bacterial strain is being used for 
the cohort in which they are enrolled. 
Masking Procedures
Because all subjects in a given cohort will receive the same inoculum suspension, masking 
procedures during the trial are not applicable.  Laboratory personnel performing assays to 
monitor host responses will be blinded to the subjects’ infection status at the time of specimen 
collection. 
Reasons for Withdrawal 
A study subject will be discontinued from participation in the study if: 
 Any clinical adverse event (AE), laboratory abnormality, intercurrent illness, or other 
medical condition or situation occurs such that continued participation in the study would not 
be in the best interest of the subject. 


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 A subject signs the consent and changes his mind about participating in the study before 
inoculation; he will be withdrawn from study participation.   
 A urethral abnormality that prevents normal insertion of the catheter is found during the 
inoculation procedure.  The subject will be treated to prevent infection from potential 
bacterial exposure during attempted inoculation and will be withdrawn from study 
participation.  A follow-up visit will be scheduled for reexamination and urine testing for N. 
gonorrhoeae infection.   
 Following inoculation, subjects will be withdrawn if they experience trauma from insertion 
of the catheter as determined by 5 or more red blood cells per high power field observed in 
urine sediment, or if they develop an infection unrelated to the study that requires antibiotic 
treatment.  Subjects who are withdrawn for urethral trauma will be treated whether known to 
be infected or not, and a follow-up visit will be scheduled for urine testing for N. 
gonorrhoeae infection, and the subject will be contacted for the 2-week final follow-up call. 
Subjects are free to withdraw from participating in the study at any time upon request. 
Handling of Withdrawals 
Occasionally, subjects fail to appear for inoculation after initially signing the informed consent 
form at screening.  For subjects who fail to appear on the day of inoculation, previously obtained 
blood and urine specimens will be destroyed.  All subjects who are inoculated, or for whom 
inoculation is attempted but unsuccessful due to a previously unrecognized anatomical 
abnormality, will be treated and followed for safety even if they decide to withdraw from the 
study or are withdrawn for any of the reasons listed above in section 5.3.3.  Subjects who are 
withdrawn after inoculation or attempted inoculation will be scheduled to return for the 1 week 
follow-up visit including the test of cure and will be contacted 2 weeks after the subject received 
treatment for the final follow-up phone call.  Withdrawn subjects may be replaced.  If the results 
of the withdrawn subject(s) would have impacted the decision to continue to enroll up to 8 
subjects in the mutant group, then the subject(s) will be replaced.  Otherwise, the withdrawn 
subject(s) will not be replaced.  Similarly, for competitive infections, the withdrawn subject(s) 
will only be replaced if their results would have impacted the decision to continue to enroll up to 
16 subjects. 
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 SMC review and recommendation; and/or at 
the discretion of DMID. 


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6 STUDY INTERVENTION/INVESTIGATIONAL PRODUCT 
N. gonorrhoeae strain FA1090, the parent strain for isogenic mutants in this study, is serum and 
streptomycin resistant and is sensitive to cefixime, ciprofloxacin and ceftriaxone.  This strain 
expresses porin serovar PIB3 and is a proline auxotroph.  All FA1090 derivatives used for 
experimental infection studies retain the antibiotic susceptibility profile of the parent without 
additional resistance markers.  The genome of strain FA1090 was sequenced with support from 
USPHS/NIH grant #AI38399, and B.A. Roe, L. Song, S. P. Lin, X. Yuan, S. Clifton, Tom 
Ducey, Lisa Lewis and D.W. Dyer at the University of Oklahoma. The GenBank accession 
number for the completed Neisseria gonorrhoeae genome is AE004868. Strain FA1090 has been 
submitted to the ATCC and given accession number ATCC 700825.   
6.1 Study Product Description 
 Acquisition 
FA1090 has been passaged repeatedly in the research laboratories at UNC since its isolation 
from a woman with cervicitis in the 1970s at UNC Hospitals.  The original inoculum stock used 
at UNC in experimental human infection studies, prepared in 1991, was an Opa-negative, 
piliated variant designated FA1090 A21.(17)  Two independent second generation stocks, A22 
and A23 (also Opa-negative and piliated) were prepared from FA1090 A21 in 1994 and 1997, 
respectively.  In December 2015, the isogenic mutant FA7537 (FA1090 mtrD) that does not 
express MtrD and the fourth generation wild type, Opa-negative, piliated variant, FA1090 A26 
were derived directly from FA1090 A25. The isogenic mutant strain to be used in the MtrD 
Study was constructed in the laboratory of Dr. William Shafer at Emory University and 
characterized for experimental human infections in the laboratory of Dr. Marcia Hobbs at the 
University of North Carolina at Chapel Hill.  FA7537 contains a deletion of the mtrD gene, made 
without leaving behind a selectable marker as previously described.(23)  Briefly, a two-gene 
cassette containing both a selectable marker (chloramphenicol acetyl transferase [CAT] 
conferring chloramphenicol resistance) and a counter selectable marker (rpsL, conferring 
streptomycin susceptibility on the naturally resistant FA1090) was cloned into the gene of 
interest and used to replace the wild-type gene on the chromosome by allelic exchange. A second 
transformation replaced the cassette-containing version of the gene with an engineered version 
with an unmarked deletion or other mutation. Importantly, susceptibility to cefixime, ceftriaxone 
and ciprofloxacin are documented for FA7537 and all N. gonorrhoeae strains for use in 
experimental infection studies. All strains will be reviewed and approved for use in the 
experimental human infection model by the Institutional Biosafety Committee (IBC) of the 
University of North Carolina at Chapel Hill.   
 Formulation, Packaging, and Labeling 
All components used to make the media and buffers used to prepare the bacterial inocula are 
made from sequestered lots whose certificates of analysis are on file.  GC agar base (used to 


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prepare solid microbiological media [GCB agar] on which bacterial inoculum strains are grown) 
and Trypticase soy broth powder (used to prepare freezer storage medium [FSM] in which 
master/working bank stocks are stored) are derived from animal sources.  We use GC agar base 
and trypticase soy broth powder only made from certified BSE–free herds from BSE-free 
countries.  Tests of sterility are also done on all media and buffers used to prepare the inoculum.  
These media and buffers are clearly marked as use only for experimental human infection and 
are stored in a cabinet separate from other laboratory reagents. 
 
A master/working bank of each individual N. gonorrhoeae strain to be used in experimental 
human infections will be prepared from individually characterized colonies grown on GCB agar, 
selected to be phenotypically Opa-negative and expressing full-length lipooligosaccharide to 
match the characteristics of previous inocula.24, 29, 30  For each strain used to prepare a 
master/working bank, susceptibility to cefixime, ciprofloxacin and ceftriaxone will be confirmed 
in the University of North Carolina Hospitals McLendon Clinical Laboratories.  Master/working 
banks will be amplified in one passage from selected colonies, suspended in freezing medium, 
and stored as multiple identical aliquots at < -70°C in a separate freezer compartment containing 
only N. gonorrhoeae for use in experimental human infection trials.  No other bacteria or 
laboratory N. gonorrhoeae strains are stored in this freezer compartment. For master/working 
banks to be used more than 1 year after initial preparation, cfu/mL will be enumerated from a 
single vial every 18 months; if the concentration of N. gonorrhoeae in the vial is <10
5 cfu/mL, a 
new master/working bank shall be generated. For mixed infections, material from a single 
master/working bank vial for each inoculum strain will be grown overnight on GCB agar plates.  
Individual suspensions will be made in sterile phosphate-buffered saline (PBS) and bacterial 
concentrations will be estimated using a spectrophotometer.  To prepare the desired mixture, 
approximately equal concentrations of the two strains will be combined in freezing medium.  
Mixed inocula will be aliquoted into single use vials labeled with the strain names and date of 
preparation and stored at < -70°C in the specified restricted freezer compartment until use.  To 
confirm the ratio of the 2 strains in the mixture and to test for the presence of contaminants, the 
contents of one vial of the working stock will be grown on GCB agar overnight, resuspended in 
PBS and enumerated by CFU.  Growth on plates will be inspected visually and using a dissecting 
microscope to confirm the presence of only N. gonorrhoeae colonies.  Gonococcal colonies will 
be analyzed by culture on selective media to distinguish the individual strains or by PCR with 
specific primers to determine the proportion of each strain in the mixture. Inoculum mixtures 
prepared and stored as described are stable, and the proportions of individual strains recovered 
from single use vials are consistent in material analyzed up to 6 months after the initial working 
stock is prepared.  A previous 50:50 mixture sampled 5 times over a 6 month period yielded a 
mean proportion of 53:47 with a standard error of 1.36.  For working stock mixtures to be used 
more than 1 year after initial preparation, the composition of the stock will be verified before use 
to prepare an inoculum for experimental use.  
 Product Storage and Stability 
Please refer to Sections 6.1.1 and 6.1.2 for storage and stability information regarding bacterial 
suspensions.  The main supply of vials containing frozen stocks of N. gonorrhoeae is kept in a 
single compartment of a freezer maintained at < -70°C.  No other bacteria or laboratory N. 
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prepare solid microbiological media [GCB agar] on which bacterial inoculum strains are grown) 
and Trypticase soy broth powder (used to prepare freezer storage medium [FSM] in which 
master/working bank stocks are stored) are derived from animal sources.  We use GC agar base 
and trypticase soy broth powder only made from certified BSE–free herds from BSE-free 
countries.  Tests of sterility are also done on all media and buffers used to prepare the inoculum.  
These media and buffers are clearly marked as use only for experimental human infection and 
are stored in a cabinet separate from other laboratory reagents. 
 
A master/working bank of each individual N. gonorrhoeae strain to be used in experimental 
human infections will be prepared from individually characterized colonies grown on GCB agar, 
selected to be phenotypically Opa-negative and expressing full-length lipooligosaccharide to 
match the characteristics of previous inocula.24, 29, 30  For each strain used to prepare a 
master/working bank, susceptibility to cefixime, ciprofloxacin and ceftriaxone will be confirmed 
in the University of North Carolina Hospitals McLendon Clinical Laboratories.  Master/working 
banks will be amplified in one passage from selected colonies, suspended in freezing medium, 
and stored as multiple identical aliquots at < -70°C in a separate freezer compartment containing 
only N. gonorrhoeae for use in experimental human infection trials.  No other bacteria or 
laboratory N. gonorrhoeae strains are stored in this freezer compartment. For master/working 
banks to be used more than 1 year after initial preparation, cfu/mL will be enumerated from a 
single vial every 18 months; if the concentration of N. gonorrhoeae in the vial is <10
5 cfu/mL, a 
new master/working bank shall be generated. For mixed infections, material from a single 
master/working bank vial for each inoculum strain will be grown overnight on GCB agar plates.  
Individual suspensions will be made in sterile phosphate-buffered saline (PBS) and bacterial 
concentrations will be estimated using a spectrophotometer.  To prepare the desired mixture, 
approximately equal concentrations of the two strains will be combined in freezing medium.  
Mixed inocula will be aliquoted into single use vials labeled with the strain names and date of 
preparation and stored at < -70°C in the specified restricted freezer compartment until use.  To 
confirm the ratio of the 2 strains in the mixture and to test for the presence of contaminants, the 
contents of one vial of the working stock will be grown on GCB agar overnight, resuspended in 
PBS and enumerated by CFU.  Growth on plates will be inspected visually and using a dissecting 
microscope to confirm the presence of only N. gonorrhoeae colonies.  Gonococcal colonies will 
be analyzed by culture on selective media to distinguish the individual strains or by PCR with 
specific primers to determine the proportion of each strain in the mixture. Inoculum mixtures 
prepared and stored as described are stable, and the proportions of individual strains recovered 
from single use vials are consistent in material analyzed up to 6 months after the initial working 
stock is prepared.  A previous 50:50 mixture sampled 5 times over a 6 month period yielded a 
mean proportion of 53:47 with a standard error of 1.36.  For working stock mixtures to be used 
more than 1 year after initial preparation, the composition of the stock will be verified before use 
to prepare an inoculum for experimental use.  
Product Storage and Stability 
Please refer to Sections 6.1.1 and 6.1.2 for storage and stability information regarding bacterial 
suspensions.  The main supply of vials containing frozen stocks of N. gonorrhoeae is kept in a 
single compartment of a freezer maintained at < -70°C.  No other bacteria or laboratory N. 
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use in experimental human infection studies are stored in this freezer compartment.  A subset of 
1-3 vials of each stock is stored in a separate freezer maintained at < -70°C in a laboratory in a 
separate building on the UNC campus as a backup in case of extended power failure in the 
primary storage location.  
 
Cefixime, ciprofloxacin, ceftriaxone, lidocaine and epinephrine are obtained from commercial 
sources and stored according to the instructions in the package inserts. 
6.2 Dosage, Preparation and Administration of Study 
Intervention/Investigational Product 
For preparation of an inoculum for an experimental infection trial, the contents of one single-use 
vial from the appropriate working stock will be dispensed onto 5 individual GCB agar plates the 
day before scheduled inoculation.  Inoculum plates will be prepared in a calibrated laminar-flow 
hood in the research laboratory.  The hood will be thoroughly disinfected before and after use.  
Plates will be streaked with a sterile microbiological loop in a pattern to yield areas of dense 
growth as well as areas of isolated colony growth on the plate after incubation. On the day of 
inoculation, The PI or designee will examine growth on the plates by eye and with a dissecting 
microscope for purity and correct colony phenotype.  Any plates containing contaminating 
bacterial growth will be discarded.  From an area with isolated colony growth, several colonies 
from each plate will be tested with the oxidase reagent; colonies must test positive (turn dark 
purple within 30 seconds) to use growth from that plate for the inoculum suspension.    Plates 
passing all pre-inoculation tests will be loaded into a candle canister for transport to the CTRC 
laboratory. 
 
When all subjects have been prepared for inoculation in the CTRC inpatient unit, working in a 
disinfected dead air box providing a circulation free environment with UV light sterilization to 
protect the product from contamination, in the CTRC Investigators’ laboratory, The PI or 
designee will prepare a bacterial suspension in sterile PBS from growth harvested with a sterile 
cotton swab from agar plates.  Plates are retained for later examination for contamination.  The 
suspension will be passed through a 1.2 µm sterile syringe filter to remove bacterial clumps 
leaving a uniform suspension of gonococci.  The optical density of the filtered suspension will be 
determined as absorbance at 550 nm wavelength (A550).  This value will be compared to 
previously determined A550 readings of filtered gonococcal suspensions for which the number 
of colony forming units (CFU)/mL are known.  The A550 reading is adjusted by adding sterile 
PBS or more filtered bacteria to achieve the desired absorbance range corresponding to the target 
concentration of CFU/mL in the tube.  The tube containing the final suspension will be delivered 
to the trial physician for inoculation.  Inoculations will be completed within 30-minutes of the 
preparation of the bacterial suspension.  A portion of the inoculum suspension is plated before 
the first and after the final subject is inoculated to rule out contamination and confirm the 
bacterial dose delivered. 
 
For inoculation, subjects recline on a hospital bed and are required to don sterile gloves 
immediately before the procedure. The pelvic area is covered with a waterproof plastic drape, 


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and the penis is extended through a hole in the drape. The skin around the urethra is cleansed 
with 70% isopropanol and allowed to dry.  Allergic reactions and other side effects of topical 
alcohol antiseptics are rare.  The individually wrapped, premoistened gauze pads are the same as 
those routinely used in the hospital to cleanse skin before venipuncture.  Up to 0.4 mL of 
bacterial suspension in phosphate-buffered saline will be delivered to the anterior urethra through 
a No.8 pediatric French catheter, inserted 5 cm into the urethra.  The PI or designee will load the 
inoculum into a dedicated pipette, and a licensed physician wearing sterile gloves, will moisten 
the catheter using sterile saline, insert the catheter and deliver the inoculum using aseptic 
technique.  Inoculation takes only a few seconds and produces minimal discomfort.  Subjects are 
instructed to apply gentle pressure to the end of the penis for 30 minutes after inoculation to 
prevent leakage of the bacterial suspension.  Subjects will be examined for signs or symptoms of 
urethritis and first-void urine will be collected daily.  Subjects will be treated with cefixime 
(single 400 mg oral dose) or ceftriaxone (250 mg IM single dose) when infection is clinically 
apparent or at the end of the inpatient portion of the trial.  On the day a subject is treated, a 
urethral swab will be collected from each subject for gram stain and gonococcal culture.  
Subjects will return within 7 days of the inpatient portion of the trial for reexamination and urine 
testing by nucleic acid amplification.  In the unlikely event of antibiotic treatment failure, 
defined by a positive test of cure, subjects will be treated with ciprofloxacin (single 500 mg oral 
dose).  
 
 
For noncompetitive infections in the MtrD Study, 10
6 CFU of FA1090 A26 (WT) or FA7537 
(∆mtrD), representing estimated ID
80, will be used for urethral inoculation.  For competitive 
infections, equivalent numbers of WT FA1090 and the isogenic mutant, in a total dose ranging 
from 10
5 - 10
6 CFU, representing estimated ID50-80, will be used for urethral inoculation.  During 
experimental trials, output bacteria will be recovered from infected subjects daily from first void 
urine and at the end of the inpatient portion of the trial from a urethral swab, and the numbers of 
gonococci in each urine specimen will be enumerated by CFU.  Strains from mixed infections 
will be distinguished by their ability to grow on culture medium without polymixin B or colistin 
(both FA1090 A26 and FA7537 will grow) and on culture medium containing polymixin B or 
colistin (only FA1090 A26 will grow).   
6.3 Modification of Study Intervention/Investigational Product for a 
Participant 
 
All participants in noncompetitive infection cohorts will receive the same inoculum containing 
WT or isogenic mutant. All participants in individual competitive infection cohorts will receive 
the same mixed inoculum at the dose indicated for the specific experimental infection trial.   
6.4 Accountability Procedures for the Study Intervention/Investigational 
Product(s) 
 
Preparation and characterization of master/working banks of individual strains and mixtures will 
be performed by Dr. William Shafer, Ms. Lorraine Balletta or Dr. Andreea Waltmann and 


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overseen by Dr. Marcia Hobbs.  Please refer to section 6.2 for accountability of inoculum 
preparation and delivery; these details remain constant for all experimental infection trials to be 
conducted under this protocol, regardless of the N. gonorrhoeae strain(s) to be included in the 
inoculum. 
6.5 Assessment of Subject Compliance with Study 
Intervention/Investigational Product  
The administration of the investigational product to subjects will be under the direct control of 
the Principal Investigator, Dr. Marcia Hobbs and the trial physician, Dr. Joseph A. Duncan. 
6.6 Concomitant Medications/Treatments 
The study population will consist of healthy 18-35 year old men.  Subjects will be excluded if 
there has been use of any antibiotics in the month prior to enrollment.  During the study, all 
subjects will be treated with cefixime (single 400 mg oral dose) or ceftriaxone (250 mg IM single 
dose) when (1) he requests treatment, regardless of signs, symptoms or positive cultures (2) 
purulent urethral discharge is observed by the examining clinician or reported by the subject or 
(3) 5 days after inoculation, regardless of infection status.  In the unlikely event of antibiotic 
treatment failure defined by a positive test of cure (which has never happened among more than 
200 infected subjects to date), subjects will be treated with ciprofloxacin (single 500 mg oral 
dose).  
 
Medications not permitted with cefixime or ceftriaxone: 
• Warfarin 
• Probenecid 
• Aspirin 
• Diuretics such as furosemide 
• Aminoglycoside antibiotics 
• Chloramphenicol 
 
Medications not permitted with ciprofloxacin: 
• Tizanidine 
• Theophylline 
• Warfarin 
• Glyburide 
• Cyclosporine 
• Probenecid 
• Phenytoin 
• Methotrexate 
• Antacids, multivitamins, and other dietary supplements containing magnesium, 
calcium, aluminum, iron or zinc 
• Caffeine-containing medications 


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• Sucralfate or didanosine chewable or buffered tablets 
 
In the event of an anaphylactic reaction to administration of the bacteria or to a study medication, 
the subject will be treated with 0.3 mg of epinephrine intramuscularly in the thigh using an 
EpiPen autoinjector.  Emergency services (911) will be called, and the subject will be transported 
to the University Hospital Emergency Room. 


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7 STUDY SCHEDULE 
7.1 Screening  
• The study is explained in detail by the study coordinator, and subjects are given the 
opportunity to read the informed consent form and ask questions.   
• If a subject agrees to participate, written informed consent is obtained for the main study and 
subjects initial a statement indicating permission for specimen storage.  Subjects will 
complete a True/False test of understanding (Appendix B) following review of the consent 
form. 
• Perform a complete physical exam. 
• Obtain medical history and concomitant medications (prescription and over-the-counter 
drugs taken 30 days prior to enrollment will be reviewed and documented). 
• Vital signs are obtained. 
• Twenty-five mL of first catch urine is collected for urinalysis and nucleic acid amplification 
testing to rule out infection with Chlamydia trachomatis and Neisseria gonorrhoeae 
(CT/NG), and Trichomonas vaginalis (TV).  Screening test results outside acceptable limits 
(as detailed in sections 5.1 and 5.2) will exclude subjects from the trial; they will be referred 
to appropriate caregivers to further investigate the reasons for results deemed clinically 
significant by the study physician. 
• Up to 30mL of blood is collected for CBC with differential (for WBC, PMN and hemoglobin 
results), CH50 determination to rule out complement deficiency, ALT to rule out liver 
impairment, creatinine to rule out renal impairment, HIV, syphilis, HBV and HCV 
serologies. If the results of CBC, serum ALT, serum creatinine, or urinalysis obtained at 
initial screening are outside acceptable limits, and the clinician judges the deviation unlikely 
to be clinically relevant, one-time repeat screening is permitted.  Positive HIV, abnormal 
CH50, repeated CBC, ALT or creatinine results outside acceptable limits will exclude 
subjects from the trial; they will be referred to appropriate caregivers to further investigate 
the reasons for results deemed clinically significant by the study physician. 
• Subjects are contacted when screening test results are available.  Subjects with positive 
results are excluded from participation in the study and referred to primary care. 
• Screening is completed within 30 days prior to the inoculation visit. 
• Subjects return to the Clinical Translational Research Center outpatient unit 2-4 days before 
enrollment and inoculation to provide a urine specimen for repeat testing for CT/NG and TV 
and for storage.  In addition, up to 20mL of blood is obtained for studies of immune 
responses to gonorrhea.  Results of the eligibility STI testing will be reviewed prior to 


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enrollment; all results must be negative to proceed with inoculation.  Subjects with positive 
results are excluded from participation in the study and referred to primary care.   
7.2 Enrollment/Baseline 
• Each experimental trial begins with enrollment and inoculation for Visit 1 on Study Day 1.  
Subjects are instructed to report to the hospital for admission to the Clinical Translational 
Research Center inpatient unit at the earliest time permitted by the hospital schedule, and 
inoculation takes place between 9:00am and noon.   
• In an individual inpatient room in the CTRC, the trial physician reviews screening medical 
history, concomitant medications, physical exam and laboratory results to verify that the 
subject meets inclusion criteria for study entry.  Results of STI testing from urine obtained 2-
4 days previously will be reviewed prior to enrollment; all results must be negative to 
proceed with inoculation.  Subjects with positive results are excluded from participation in 
the study and referred to primary care.  Informed consent statements are reviewed explaining 
procedures and risks.  Subjects are given the opportunity to withdraw if they do not accept 
the risks of the study.  Subjects who agree to proceed with participation in the study will sign 
and date the study consent form.  Subjects are offered copies of the signed consent form.  
 
• Vital signs are obtained, and the study physician’s assistant, research nurse or trial physician 
conduct a targeted physical exam including brief reexamination of the genitals to exclude any 
new or previously unrecognized urethral abnormalities.  
 
• For inoculation, subject reclines on a hospital bed and is required to don sterile gloves 
immediately before the procedure. The pelvic area is covered with a waterproof plastic drape, 
and the penis is extended through a hole in the drape. The skin around the urethra is cleansed 
with 70% isopropanol and allowed to dry.  Bacterial suspensions in phosphate-buffered 
saline are delivered to the anterior urethra through a No.8 pediatric French catheter, inserted 
5 cm into the urethra.  A licensed physician wearing sterile gloves, will moisten the catheter 
using sterile saline, insert the catheter and deliver the inoculum using aseptic technique.  
Inoculation takes only a few seconds and produces minimal discomfort.  The subject is 
instructed to apply gentle pressure to the end of the penis for 30 minutes to prevent leakage 
of the inoculum.  Subjects may sign out of the research unit on the day of inoculation, no 
sooner than 45 minutes after the procedure.  Any AE will be assessed prior to discharge from 
the clinic. 
 
7.3 Follow-up  
• Each day, up to 5 days after inoculation (Visits 2-6 on Study Days 2-6), the subject 
undergoes a targeted physical exam in the inpatient ward of the clinical research center by a 
study physician, research nurse or physician’s assistant, usually between 6:30am and 7:30am 
for signs or symptoms of urethritis, review of adverse events, and a first-void urine specimen 


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of approximately 50 mL is collected for gonococcal culture, urine dipstick analysis, 
microscopic examination of urine sediment for PMNs as an indicator of urethral 
inflammation, and for storage.  
• Study personnel will review subject’s medical history and medications. 
• Daily vital signs are obtained in the morning, typically between 6:00am and 8:00am, by 
CTRC nursing staff and include blood pressure, heart rate, respiration rate and temperature.  
In the event abnormal values (indicated in Table 1 below) are obtained, the trial physician 
will be called and s/he will use good clinical judgment to ensure appropriate treatment. 
 
Table 1.  Abnormal vital signs 
 
Vital sign 
Value resulting in call to physician 
Blood pressure 
Diastolic > 90 
Heart rate 
Pulse > 130 
Respiration rate 
> 25 
Temperature 
> 100.6 °F (> 38.1 °C) 
• The subject is treated if (1) he requests treatment regardless of signs, symptoms or positive 
cultures (2) purulent urethral discharge is observed by the examining clinician or reported by 
the subject or (3) 5 days after inoculation, regardless of infection status.  Treatment is a 
single observed 400 mg oral dose of cefixime.  If he cannot swallow the oral pill, he will be 
treated with a single intramuscular dose of ceftriaxone (250 mg). Before antibiotic treatment 
on Day 6, or the day on which a subject is treated during the trial, a urethral swab and urine 
will be collected, and up to 20 mL of blood will be collected for studies of host immune 
responses during infection. Subjects will be observed for approximately 20 minutes after 
administration of study antibiotics.  Any AE will be assessed prior to discharge from the 
clinic. 
• Subjects who do not require treatment based on the criteria above may leave the CTRC ward 
as needed during the study on Days 1-6, provided they return to give specimens, identify 
their location when they are not on the ward, and stay overnight on the ward.  In most 
instances, subjects will be able to leave the ward by 7:15am and return in the evening with a 
9:00pm curfew to arrive back at the ward.  Subjects will carry a cell phone so we can reach 
them at all times, and especially if they violate the agreed curfew.  If a subject is not on the 
ward by 9:00pm, CTRC research staff will contact him and instruct him to return 
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7.4 Final Study Visit 
• Subjects return to the outpatient unit of the CTRC 3 to 7 days following antibiotic treatment 
(Visit 7, Study Day 5-13, depending on how soon after inoculation an individual subject is 
treated and released from the clinical research unit) for re-examination by the trial clinician 
and urine testing for N. gonorrhoeae by nucleic acid amplification, and for compensation.  At 
this visit, the subject will receive a targeted physical examination, review of medical history 
and medications, review of symptoms and Adverse Events, and vital signs will be obtained.  
Specimens obtained at this visit will include up to 20 mL of blood and 50 mL of first-void 
urine for urinalysis, CT/NG testing, and storage for studies of host immune responses post-
infection.   
• A final follow-up call will be conducted 7-10 days after the follow-up visit in the outpatient 
unit (Visit 8, Study Day 12-23).  Subjects will be queried about symptoms or other problems 
potentially related to participation in the trial. 
7.5 Early Termination Visit 
• If a subject elects to terminate the study early following inoculation, he will receive treatment 
as described above, and follow-up Visit 7 for re-examination, test of cure and compensation 
will be scheduled, and the final follow-up call for Visit 8 will be conducted. 
7.6 Unscheduled Visit  
• During the trial, subjects receive a scheduled examination first thing in the morning, typically 
between 6:30am and 7:30am.  Subjects are instructed to call and return to the research unit if 
they experience symptoms at anytime they are away from the CTRC.  Nursing staff are 
available 24 hours/day in the inpatient unit and will page the on-call study clinician, who will 
call the subject to discuss the severity of symptoms and meet the subject in his inpatient room 
on the research ward if needed for evaluation, specimen collection and treatment as described 
above in section 7.3.  Subjects who choose to remain in the unit can directly contact the nurse 
on duty.  For any such unscheduled, but not unanticipated event, the nurse on duty pages the 
study clinician, who contacts the subject as soon as possible and sees the subject in the 
CTRC as needed.   
   


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8 STUDY PROCEDURES/EVALUATIONS 
8.1 Clinical Evaluations 
At screening, a standard medical history and complete physical examination will be performed 
and documented by the study physician’s assistant in an outpatient room at the CTRC.  
Specifically, information will be obtained to exclude individuals with any of the following: 
• History of STIs including gonorrhea, chlamydia, syphilis, HIV, hepatitis B & C 
• History of bleeding diathesis 
• History of seizures (due to reports of seizures with ciprofloxacin) 
• History of cancer, except basal cell carcinoma of the skin more than 5 years ago 
• History of drug abuse  
• History of psychiatric disorders, except depression controlled by medication 
• History of genitourinary surgery 
• Any known immunodeficiencies including complement deficiency, antibody deficiency, 
chronic granulomatous disease or HIV infection 
• Heart murmur or heart disease 
• Clinically relevant abnormalities on physical exam 
• Abnormal urine leukocyte esterase or WBC 
• Protein or RBC in urine outside allowable limits 
• WBC count, ANC, or hemoglobin level outside allowable limits 
• Serum creatinine or ALT levels outside allowable limits 
• Positive screening test for CT, NG or TV 
• Positive serology for HIV, syphilis, HBV or HCV 
• Anatomic abnormality of the urinary tract 
• Antibiotic treatment in the past 30 days or azithromycin in the past 60 days 
• Chemotherapy within the past year 
• Current steroid use, except for topical application 
• Allergy to penicillin, cefixime, ceftriaxone, ciprofloxacin or to lidocaine 
• Treatment with medications that are contraindicated with cefixime, ceftriaxone or 
ciprofloxacin and that cannot be withheld for the single doses given in this study. 
 
Two to four days before enrollment, subjects will provide a urine specimen for CT/NG and TV 
testing to confirm eligibility.  At the enrollment visit and before inoculation, results from the 
eligibility testing will be reviewed; subjects with any positive test will be excluded.  Provided all 
STI test results are negative, a brief urethral examination will be performed before inoculation. 
 
For five days after inoculation, subjects will be examined daily.  The following will be solicited 
during the targeted genital examination: 
 
Subjective symptoms: 
• Tingling 
• Irritation or itching 
• Dysuria 


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• Urinary frequency 
• Testicular pain 
• Feverish 
• Chills 
• Malaise 
• Diarrhea 
• Rash 
 
Objective signs: 
• Urethral discharge  
 
A first-void urine specimen will be collected.  All subjects will receive antibiotic treatment 
before discharge from the inpatient portion of the study.  Subjects are treated if (1) he requests 
treatment regardless of signs, symptoms or positive cultures (2) purulent urethral discharge is 
observed by the examining clinician or reported by the subject or (3) 5 days after inoculation, 
regardless of infection status.  On the last day of the inpatient portion of the trial, or the day on 
which a subject receives treatment for gonococcal infection, a urethral swab will be obtained.  
Subjects will be observed for approximately 20 minutes following administration of study 
antibiotics. 
 
Subjects return to the CTRC within one week for re-examination by a physician or physician’s 
assistant licensed to treat gonococcal infections and their complications, and a urine CT/NG 
NAAT test of cure.  The test of cure is a nucleic acid amplification test.  In the unlikely event of 
a positive test of cure, we will obtain a urethral specimen for culture to enable antibiotic 
susceptibility testing.   
8.2 Laboratory Evaluations 
 Clinical Laboratory Evaluations 
• Serum CH50 test for complement deficiency, creatinine, ALT and HIV, syphilis, HBV and 
HCV serologies and CBC at screening 
• Urine-based nucleic acid amplification testing for CT/NG and TV and urinalysis at 
screening; urine CT/NG and TV tests 2-4 days prior to enrollment; urine CT/NG test and 
urinalysis at 1 week follow-up (Day 5-13). 
 Special Assays or Procedures 
• Daily urine sediment culture for N. gonorrhoeae to monitor infection (Study Days 2-6).  
Bacteria isolated from urine cultures will be enumerated, and individual colonies will be 
stored for research testing to compare numbers and the genotypes and phenotypes of output 
compared to input organisms. 


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• Daily urine dipstick analysis for hematuria (Study Days 2-6). 
• Daily microscopic examination of urine sediment and quantitation of WBCs to monitor 
inflammation (Study Days 2-6).   
• Stored urine (collected 2-4 days prior to enrollment, on Study Days 2-6, and at 1 week 
follow-up [Study Days 5-13]) and blood (collected 2-4 days prior to enrollment, on day of 
antibiotic treatment, and at 1 week follow-up [Study Days 5-13]) from subjects who provide 
permission for stored specimens will be used to measure cytokines, chemokines and other 
molecules that may mediate host immune responses to gonococcal infection.  Levels of 
individual cytokines will be assessed using commercially available ELISAs.  Multiplexed 
cytokine analysis will be measured using slide-based anticytokine antibody arrays or 
microparticle based arrays.  Activation of inflammatory cell death programs will be assessed 
by staining with a fluorescent stain excluded by viable cells followed by quantitation of 
staining by flow cytometry.  Assays of immunological parameters are purely for pursuit of 
research objectives, and results will not be used for clinical purposes. 
 Specimen Preparation, Handling, and Shipping 
• Approximately 30 mL of blood will be obtained at screening.  Screening labs will be sent to 
the NC Hospitals McLendon Clinical Laboratories for CH50, CBC, ALT, creatinine and 
HIV, syphilis, HBV and HCV serology tests.  Blood and urine obtained at the eligibility 
testing visit and at followup will be sent to Dr. Hobbs’ Laboratory where specimens will be 
processed and stored at < -70°C according to laboratory SOPs.   
• Urine obtained at screening, before enrollment and at followup will be sent to the NC 
Hospitals McLendon Clinical or UNC CFAR VIM Core Laboratories for nucleic acid 
amplification testing for C. trachomatis, N. gonorrhoeae and T. vaginalis.  Excess urine will 
be transferred to Dr. Hobbs’ Lab, aliquoted and stored frozen at < -70°C. 
• Daily urine specimens will be processed by Dr. Hobbs or other study personnel in the 
investigators’ laboratory at the CTRC.  Urine dipstick analysis will be performed on whole 
urine (8-10 mL).  This volume will then be centrifuged in the investigators’ lab at the CTRC, 
and sediment will be examined microscopically to enumerate WBCs.  The remaining urine 
from the original 50-mL specimen will be centrifuged separately.  The supernatant from the 
spun urine will be aliquoted and stored frozen at < -70°C in Dr. Hobbs’ Lab.  The urine 
sediment will be inoculated onto GCB agar plates for N. gonorrhoeae culture; inoculated 
plates will be loaded into candle jars for transport from the CTRC to Dr. Hobbs’ Lab.  
Individual N. gonorrhoeae colonies isolated from urine cultures (up to 100 per subject per 
day) will be stored frozen at < -70°C in Dr. Hobbs’ Lab. 


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8.2.3.1 Instructions for Specimen Preparation, Handling, and Storage 
• Specimens obtained from subjects who deny permission for long-term storage will be 
indicated with an easily visible sticker marked NO STORAGE.  Such specimens will be 
destroyed at the conclusion of the study.   
SOPs for specimen preparation, handling and storage according to the information in Section 
8.2.3 will be submitted to the Clinical Project Manager following DMID protocol approval. 
8.2.3.2 Specimen Shipment 
No shipping to external facilities is required for this study.  Specimens will be transported by 
hospital or study personnel in appropriate biosafety containers/bags. 


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9 ASSESSMENT OF SAFETY 
9.1 Specification of Safety Parameters 
To minimize risks, the study is designed to enroll healthy male subjects and to exclude 
individuals with a complement deficiency, renal or liver impairment, or allergy to study 
medications and women, who cannot safely be infected due to potential complications from 
ascendant gonococcal infection.  The bacterial doses range from 10
5 to 10
6 CFU of N. 
gonorrhoeae, corresponding to the approximate ID
50 and ID
80, respectively, for the parent N. 
gonorrhoeae strain FA1090.  These doses are similar to or smaller than typical exposures in 
naturally-acquired gonococcal infection.   
Delivery of 106 CFU of wild type FA1090 to the anterior urethra results in infection 
approximately 80% of the time with symptoms of urethritis manifesting 3 to 4 days after 
inoculation.     
Careful screening and observation during participation, overnight stays in the CTRC, prompt 
treatment of symptoms of gonococcal infection and treatment prior to discharge from the study 
are all designed to minimize the possibility of complications.  Subjects will be examined daily by 
the study physician or the study physician’s assistant first thing in the morning (typically 
between 6:30am and 7:30am), when signs or symptoms of urethral discharge or dysuria are most 
likely to be observed.  In addition, study personnel are available for evaluation, specimen 
collection and treatment if subjects experience symptoms and require treatment any time during 
the day or night after the regularly scheduled morning examination.  A study physician is on 
call at all times during experimental infection trials.  
Infection will be terminated by treating the subject with a single observed 400 mg oral dose of 
cefixime.  If he cannot swallow the oral pill, he will be treated with a single intramuscular dose 
of ceftriaxone (250 mg). Treatment will be given after one of the following endpoints has been 
achieved: (1) subject requests treatment regardless of signs, symptoms or positive cultures (2) 
purulent urethral discharge is observed by the examining clinician or reported by the subject or 
(3) 5 days after inoculation, regardless of infection status.  Subjects will return within 7 days of 
antibiotic treatment for reexamination and a test of cure. In the unlikely event of antibiotic 
treatment failure, defined by a positive test of cure (which has never happened among more than 
200 infected subjects to date), subjects will be treated with a single observed 500 mg oral dose of 
ciprofloxacin.  In the event of an anaphylactic reaction to administration of the bacteria or to a 
study medication, the subject will be treated with 0.3 mg of epinephrine intramuscularly in the 
thigh using an EpiPen autoinjector.  Emergency services (911) will be called, and the subject will 
be transported to the University Hospital Emergency Room.  
 
 
 


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9.2 Methods and Timing for Assessing, Recording, and Analyzing Safety 
Parameters 
 Adverse Events   
All AEs that occur from inoculation through the 2 week final follow-up phone call (Study Days 
12-23) will be reported.  At each scheduled visit, AEs will be assessed and any AEs identified 
during this period will be reported.  
The Investigator is responsible for reporting all AEs that are observed or reported during the 
study, regardless of their relationship to study product. 
 
Adverse Event:  ICH E6 Good Clinical Practice Guidelines defines an Adverse Event (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 medicinal (investigational) product.  
The occurrence of an adverse event may come to the attention of study personnel during study 
visits and interviews or by a study recipient presenting for medical care, or upon review by a 
study monitor.    
All AEs including local and systemic reactions not meeting the criteria for “serious adverse 
events” should be captured on the appropriate CRF.  Information to be collected includes event 
description, time of onset, clinician’s assessment of severity, relationship to study product 
(assessed only by those with the training and authority to make a diagnosis, which would include 
MD, PA, Nurse Practitioner, DO, or DDS), and time of resolution/stabilization of the event.  All 
AEs occurring while on study must be documented appropriately regardless of relationship.  All 
AEs will be followed to adequate resolution.  
Any medical condition that is present at the time that the patient is screened should be considered 
as baseline and not reported as an AE.  However, if it deteriorates at any time during the study, it 
should be recorded as an AE. 
 
All adverse events will be graded for severity and relationship to study product.  
 
Severity of Event:  All adverse events will be assessed by the investigator according to the 
DMID Toxicity Table (May 2015) as follows: 
  
• Mild: events do not interfere with the patient’s daily activities. 
• Moderate: events may cause some interference with activity not requiring medical 
intervention. 
• Severe: events prevent daily activity and require medical intervention.  
 


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Changes in the severity of an AE should be documented to allow an assessment of the duration 
of the event at each level of intensity to be performed.  Adverse events characterized as 
intermittent require documentation of onset and duration of each episode. 
 
Relationship to study products:  The investigator’s assessment of an AE
's relationship to 
study procedure or drug 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.  All adverse events must have their 
relationship to study product assessed using the following 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. 
 
Relatedness to study product must specify whether to N. gonorrhoeae inoculum, cefixime, 
ceftriaxone or ciprofloxacin. 
Expected AEs are those that occur during or following the study procedures (i.e. urethral 
inoculation, discomfort from collecting specimens, bruising from blood draw) or study 
medication administration which are outlined in the potential risks of the study medication 
package inserts and have no other definitely related explanation. Unexpected AEs are those 
events that occur and are not listed in the package insert for the study medication or events that 
are not the usual side effects associated with the study procedures. 
 Reactogenicity (for Vaccine Studies and Some Therapeutic Trials) 
Not applicable 
 Serious Adverse Events 
Suspected adverse reaction means any adverse event for which there is a reasonable possibility 
that the drug caused the adverse event.  For the purposes of IND safety reporting, “reasonable 
possibility” means there is evidence to suggest a causal relationship between the drug and the 
adverse event.  Suspected adverse reaction implies a lesser degree of certainty about causality 
than adverse reaction, which means any adverse event caused by a drug. 
Unexpected adverse event or unexpected suspected adverse reaction.  An adverse event or 
suspected adverse reaction is considered “unexpected” if it is not listed in the investigator 


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brochure or is not listed at the specificity or severity that has been observed or, if an investigator 
brochure is not required or available, is not consistent with the risk information described in the 
general investigational plan or elsewhere in the current application as amended.  For example, 
under this definition, hepatic necrosis would be unexpected (by virtue of greater severity) if the 
investigator brochure referred only to elevated hepatic enzymes or hepatitis.  Similarly, cerebral 
thromboembolism and cerebral vasculitis would be unexpected (by virtue of greater specificity) 
if the investigator brochure listed only cerebral vascular accidents.  “Unexpected” as used in 
this definition, also refers to adverse events or suspected adverse reactions that are mentioned in 
the investigator brochure as occurring with a class of drugs or as anticipated from the 
pharmacological properties of the drug, but are not specifically mentioned as occurring with the 
particular drug under investigation. 
21 CFR 312.64 Investigator reports 
Safety reports:  An investigator must immediately report to the sponsor any serious adverse 
event, whether or not considered drug related, including those listed in the protocol or 
investigator brochure and must include an assessment of whether there is a reasonable 
possibility that the drug caused the event.  Study endpoints that are serious adverse events (e.g., 
all cause mortality) must be reported in accordance with the protocol unless there is evidence 
suggesting a causal relationship between the drug and the event (e.g., death from anaphylaxis).  
In that case, the investigator must immediately report the event to the sponsor.  The investigator 
must record non serious adverse events and report them to the sponsor according to the 
timetable for reporting specified in the protocol. 
Some protocols may list events specific to the protocol that they want reported as serious with 
expedited reporting.  An example might be for a maternal immunization protocol before 
eclampsia or preterm delivery. 
Serious Adverse Event (SAE):  
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 
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• 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. 
 
 
 
Relationship to study products:  SAEs will be assessed regarding relationship (Related or 
Not Related, expected/unexpected) to study procedures or antibiotics used in the study.  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. 
 
Relatedness to study product will specify whether to N. gonorrhoeae inoculum, cefixime, 
ceftriaxone or ciprofloxacin. 
Expected SAEs are those that occur during or following the study procedures (i.e. Urethral 
inoculation, discomfort from collecting specimens, bruising from blood draw) or study 
medication administration, which are outlined in the potential risks of the study medication 
package inserts and have no other definitely related explanation. Unexpected SAEs are those 
events that occur that are not listed in the package insert for the study medication or events that 
are not the usual side effects associated with the study procedures. 
 


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 Procedures to be Followed in the Event of Abnormal Laboratory Test Values or 
Abnormal Clinical Findings 
Abnormal urine dipstick values will be reported to the study physician who will follow through 
with appropriate clinical testing, care and treatment. 
 
9.3 Reporting Procedures 
 Serious Adverse Events 
Adverse Events 
Adverse Events including local and systemic reactions not meeting the criteria for “serious 
adverse events” will be captured in the investigator’s records for the subject.  Information to be 
collected includes event description, time of onset, investigator assessment of severity, 
relationship to study product, time of resolution of the event, seriousness, and outcome.  All 
adverse events occurring while on study must be documented appropriately regardless of 
relationship. All AEs will be followed to adequate resolution.  
 
Any medical condition that is present at the time that the patient is screened should be considered 
as baseline and not reported as an AE.  However, if it deteriorates at any time during the study it 
should be recorded as an AE. 
 
Serious Adverse Events 
 
All serious adverse events will be: 
 
• recorded on the appropriate serious adverse event case report form 
• followed through resolution by a study physician 
• reviewed by a study physician 
 Regulatory Reporting for Studies Conducted Under DMID-Sponsored IND 
 
 
All SAEs will be submitted 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 


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Other supporting documentation of the event may be requested by the DMID Pharmacovigilance 
Group and should be provided as soon as possible. 
The DMID medical monitor and clinical protocol 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 investigator becomes aware of an SAE that is 
suspected to be related to study product, the investigator will report the event to the DMID 
Pharmacovigilance Group. 
 Regulatory Reporting for Studies Not Conducted Under DMID-Sponsored IND 
Not applicable 
 Other Adverse Events (if applicable) 
The occurrence of transmission of N. gonorrhoeae to a non-study subject will be reported.  This 
has not occurred in prior studies.  If an enrolled subject were to report having had sex during the 
timeframe relevant to study activities, the partner(s) would be referred to the UNC infectious 
diseases clinic, evaluated and given empiric treatment.  Specimens would be obtained from the 
partner(s) to determine whether the infecting organism matched the inoculated subject’s 
infection. The partner would be asked to provide informed consent to undergo non-standard of 
care procedures to evaluate the infection.  
The occurrence of N. gonorrhoeae inoculum contamination and, if applicable, clinical events 
resulting from contamination, microbiological failure of cefixime or ceftriaxone treatment post 
infection, and response to ciprofloxacin in treatment failures will be reported to DMID 
Pharmacovigilence to be forwarded to DMID and the SMC and to the on-site ISM within 2 
weeks of occurrence.  If these events meet criteria for serious adverse event, they will be 
reported on an SAE form according to SAE reporting procedures. 
• Site SAEs will be reported to DMID Pharmacovigilance according to the protocol and, 
separately, to the on-site ISM.  DMID Pharmacovigilance will forward SAEs to DMID and, 
separately, to the DMID SMC Coordination staff for forwarding to the SMC. 
• Any serious and unexpected (not listed in the package insert) adverse events considered to be 
related to licensed products that are administered in the course of the study (cefixime, 
ceftriaxone, ciprofloxacin, lidocaine, epinephrine) will be reported via DMID 
Pharmacovigilance to the FDA and separately to the on-site ISM. DMID Pharmacovigilance 
will forward SAEs to DMID and, separately, to the DMID SMC Coordination staff for 
forwarding to the SMC. 


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 Reporting of Pregnancy 
Not applicable 
9.4 Type and Duration of Follow-up of Subjects after Adverse Events 
Subjects who experience an adverse event will be followed until symptoms resolve or stabilize.  
Follow-up will continue through the duration of scheduled study visits.  If a subject has 
completed the required study visits and symptoms persist, the subject will be monitored by 
telephone or clinic visit until the adverse event resolves or stabilizes.  
9.5 Halting Rules 
The risk of major complications to subjects in the experimental setting is remote based on the 
natural history of gonorrhea and previous human experimentation.  However, the study will be 
monitored by a Safety Monitoring Committee (SMC) to ensure safety and proper performance of 
the study.  If concerns affecting subject safety or data integrity are identified by the SMC, no 
new subjects will be enrolled, but protocol procedures will continue, to ensure the safety of the 
subjects who have been inoculated.  The trial will be halted (at least temporarily) if at any time 
during the study: 
1. One SAE related to study procedure or study product(s) 
2. Three or more severe AEs of the same type (described by the same MedDRA 
SOC and Preferred Term) related to study procedure or study product(s) 
3. One antibiotic treatment failure (so that susceptibility of the isolate may be 
evaluated) 
4. Documented contamination of inoculum 
5. A late, or systemic complication of gonococcal infection including any of the 
following is reported: 
a. recovery of the infecting N. gonorrhoeae strain(s) from a normally sterile 
extragenital site 
b. petechial or pustular acral skin lesions  
c. asymmetric polyarthralgia  
d. tenosynovitis  
e. oligoarticular septic arthritis 


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Any events that trigger a halt of the study will be reviewed by the SMC, and SMC approval will 
be required before restart.  
9.6 Safety Oversight (ISM plus SMC) 
Safety oversight will be under the direction of an Independent Safety Monitor (ISM) and a 
Safety Monitoring Committee (SMC). The ISM will evaluate individual and cumulative 
participant data when making recommendations regarding safe continuation of the study at the 
time of each SMC meeting.  ISM safety concerns are noted in the SMC report.  In addition, the 
ISM will provide an independent written assessment to DMID for each SAE. 
 
The primary responsibility of the SMC is to monitor participant safety.  The SMC will review 
the research protocol and plans for data and safety monitoring. The committee will evaluate the 
progress of trials, including periodic assessments of data quality and timeliness, participant 
recruitment, accrual and retention, participant risk versus benefit, and other factors that can affect 
study outcome.  Monitoring will also consider factors external to the study when interpreting the 
data, such as scientific or therapeutic developments that may have an impact on the safety of the 
participants or the ethics of the study. The committee will protect the confidentiality of the trial 
data and the results of monitoring. 
 
The SMC will convene at the end of testing all subjects for a given N. gonorrhoeae mutant and 
on an ad hoc basis should immediate safety concerns arise; a simple majority of the SMC will be 
considered a quorum. In addition, the SMC will formally meet or electronically review the safety 
data from up to 16 evaluable subjects enrolled in noncompetitive infection groups and 
recommend the site to proceed with testing subjects in a competitive group for that mutant or to 
stop.  Evaluable subjects are those who (1) receive a dose of N. gonorrhoeae within 1 log
10 of the 
intended dose and (2) reach an objective study endpoint (urethral discharge or day 6).  When the 
investigators determine that a stopping point has been reached for an individual mutant under 
investigation, the SMC will meet to review adverse events and interim analyses, as summarized 
in the table below.  Additional subjects beyond the number proposed in the schematic for an 
individual mutant will not be inoculated without approval of the SMC.   
  
Cohort Stage 
Milestone/Trigger 
Type of Review 
  Noncompetitive infections 
4-8 mutant and up to 8 WT 
subjects  
SMC safety review and 
interim analyses 
  Competitive infections (if 
necessary) 
Up to 16 subjects  
SMC safety review and 
final analysis 
Either stage 
Presence of safety concern 
Ad hoc SMC safety 
review 
 


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10 CLINICAL MONITORING 
10.1 Site Monitoring Plan 
Site monitoring will be conducted to ensure human subject protection, study procedures, 
laboratory procedures, study intervention administration, and data collection processes are of 
high quality and meet sponsor, GCP/ICH, and regulatory guidelines, and that the study is 
conducted in accordance with the protocol and sponsor SOPs.  DMID, the sponsoring agency, or 
its designee will conduct site monitoring visits as detailed in the 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, case report forms, informed consent forms, medical and 
laboratory reports and protocol compliance.  Study monitors will meet with investigators to 
discuss any problems and actions to be taken and document visit finding and discussions. 


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11 STATISTICAL CONSIDERATIONS 
11.1 Study Hypotheses 
We hypothesize that key virulence determinants involved in N. gonorrhoeae resistance to innate 
immunity are essential for infection in the male urethra.  We predict that mutations abolishing 
expression of these virulence determinants will eliminate or significantly reduce gonococcal 
infectivity or the ability to induce inflammation in an infected individual, thus identifying 
potential vaccine candidates. 
This is an experimental infection model study designed to test the hypothesis that the gonococcal 
MtrD protein is required to establish infection in the male urethra.  To compare the ability of 
mutant gonococci to infect the male urethra, we will first inoculate subjects with WT or an 
isogenic mutant strain of N. gonorrhoeae in noncompetitive infection studies. The null 
hypothesis for this comparison is: the proportion of subjects that become infected by day 6 with 
the mutant strain is equal to the proportion of subjects that become infected by day 6 with the 
WT strain.  The one-sided alternative hypothesis for this comparison is:  the proportion of 
subjects that become infected by day 6 with the mutant strain is less than the proportion of 
subjects that become infected by day 6 with the WT strain.  
For isogenic mutants that do not demonstrate statistically significant differences in infectivity, 
we will conduct competitive infection studies to determine whether the mutant exhibits a fitness 
defect compared to WT. Competitive infections, initiated with equivalent numbers of WT and an 
isogenic mutant, allow demonstration of a difference in fitness between two strains using fewer 
subjects.   
  
For competitive infections initiated with inocula containing a mixture of WT and isogenic 
mutant strains, the competitive index (CI) of the mutant compared to WT is the measure of 
relative fitness to determine the primary outcome.  The precise proportion of each strain in the 
inoculum is determined for each experimental cohort.  The CI is defined as the ratio of colony-
forming units (cfu) of the two strains recovered from urine cultures on the day of treatment 
(output) compared to the ratio of strains in the inoculum (input) using the equation: CI = mutant 
cfu(output)/wild-type cfu(output) ÷ mutant cfu(input)/wild-type cfu(input).   
 
The null hypothesis for this comparison is: The ratio of colony-forming units of the mutant strain 
to those of the WT strain on the day of treatment is equal to the ratio of colony-forming units of 
the mutant strain to those of the WT strain in the inoculum, indicating no difference in fitness 
between the two strains during urethral infection. 
 
The one-sided alternative hypothesis for this comparison is:  The ratio of colony-forming units of 
the mutant strain to those of the WT strain at the time of treatment is less than the ratio of 
colony-forming units of the mutant strain to those of the WT strain in the inoculum, indicating 
the mutant has a fitness defect compared to the wild-type.   
 


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The hypotheses are formally tested at the interim analysis and again at the final analysis.  Thus a 
type I error rate of 0.025 is used for each hypothesis at the interim and final analyses to maintain 
an overall rate of 0.05 for each hypothesis. Because of the exploratory nature of the trial, no 
corrections for multiplicity will be performed. 
11.2 Sample Size Considerations 
 
Due to the experimental nature of the study, the small sample sizes of the individual cohorts 
were chosen based on logistical and feasibility considerations.   
 
For noncompetitive infections initiated with inocula containing an individual GC strain, the 
proportion of subjects that become infected is the measure of infectivity.  Sample size 
considerations for noncompetitive infections are summarized in Table 2 below.  Subjects will be 
enrolled in cohorts of up to 8.  We will compare infectivity of WT N. gonorrhoeae and the 
isogenic mutant using Fisher’s Exact Test.  The interim analysis (see also Section 11.3) will 
include results from up to 8 subjects inoculated with the WT N. gonorrhoeae strain only and at 
least 4 subjects inoculated with the isogenic mutant only.  Assuming the true probabilities of 
infection are 80% with wild-type and 4% with mutant inocula, 8 men per group provides 89% 
power to detect a difference using a 1-sided Fisher exact test and significance level of 0.025.  
 
Table 2.  Sample size considerations for noncompetitive infection studies 
Infection Status 
P-value1 
Number of 
Subjects 
Needed for 
analysis 
Wild-Type 
Mutant 
Yes 
No 
Yes 
No 
8 
0 
0 
4 
0.002 
12 
1 
3 
0.018 
2 
2 
0.091 
 
2 
6 
0.003 
16 
3 
5 
0.013 
4 
4 
0.038 
 
7 
1 
0 
4 
0.010 
12 
1 
3 
0.066 
 
1 
7 
0.005 
16 
2 
6 
0.020 
3 
5 
0.059 
 
6 
2 
0 
4 
0.030 
 
0 
8 
0.003 
16 
1 
7 
0.020 
2 
6 
0.066 
 
5 
3 
0 
4 
0.071 
 
0 
8 
0.012 
16 
1 
7 
0.059 
 
1P-value is from the one-sided Fisher’s Exact Test with alpha = 0.025 


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For competitive infections with mixed inocula, sample sizes are determined by the expected 
standard deviation of the mean proportion of recovered WT gonococci.  Because calculations are 
based on the numbers of bacteria, constituting larger sample sizes than infectious dose 
calculations based on the number of infected subjects, mixed infections can provide a more 
accurate indication of attenuation using fewer subjects than traditional comparisons of infectivity 
using separate, inocula with individual strains (24).  Note that competitive infections will not 
necessarily be conducted for isogenic mutants that demonstrate statistically significant 
differences in the primary and secondary outcomes specified for noncompetitive infections.  The 
total number of subjects specified in the current protocol (n = 32) refers to the maximum number 
of participants to be prospectively enrolled in noncompetitive and competitive studies for the 
MtrD mutant.  The total of 32 prospectively enrolled subjects for the combined MtrD mutant 
studies provides sufficient sample sizes for both noncompetitive and competitive analyses as 
described. 
11.3 Planned Interim Analyses (if applicable) 
 Infectivity Outcomes Review 
Interim analyses for noncompetitive infection studies will include results from up to 8 evaluable 
subjects inoculated with the WT N. gonorrhoeae strain and at least 4 evaluable subjects 
inoculated with the isogenic mutant (See Table 2 above). For interim analyses of noncompetitive 
infections, data for WT may include results from the current working bank, regardless of the 
proximity in time of the specific isogenic mutant group. See Section 5.3.4 for handling of 
withdrawals.  
At the interim analysis, the following decision rule for the noncompetitive analysis will be used: 
• If the p-value from Fisher’s Exact Test is less than 0.025, we will consider stopping the 
mutant group.  
• If the p-value from Fisher’s Exact Test is greater than 0.025 and the p-value will still be 
greater than 0.025 regardless of the results from additional subjects in the mutant group, we 
may consider closing enrollment for the noncompetitive infection study early for futility and 
proceeding with the competitive infection study. 
• If the p-value from Fisher’s Exact Test is greater than or equal to 0.025, we will continue to 
enroll until evaluable data are obtained from at least 7 subjects in the mutant group.     
If additional subjects are enrolled in the mutant group, a second interim analysis will include 
results from all evaluable subjects in the mutant and WT groups.  At the second interim analysis, 
the following decision rule for the noncompetitive analysis will be used: 
• If the p-value from Fisher’s Exact Test is less than 0.025, we will consider stopping studies 
for the isogenic mutant.  


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• If the p-value from Fisher’s Exact Test is greater than or equal to 0.025, we will conclude that 
a difference in infectivity cannot be determined in noncompetitive experimental human 
infections.     
 
If the infectivity of the two strains is not significantly different at that point, we will proceed with 
competitive infections to determine the relative fitness of the isogenic mutant. Interim analyses 
for competitive infections will be conducted after at least 5 subjects have been inoculated with a 
specific mixture of WT and mutant.  The following decision rule will be used for the interim 
analysis: 
• If the p-value from the sign test of the competitive index is less than 0.025, we will consider 
stopping the competitive infection group 
• If the p-value from the sign test of the competitive index is greater than or equal to 0.025, we 
will continue to enroll up to 16 subjects. 
 Safety Review  
 
The SMC will convene to review subject safety as described in section 9.6.  Safety outcome 
measures include adverse events, protocol deviations and any unexpected events that occurred 
since the previous review.  Primary and secondary study outcomes for subject groups enrolled to 
test an individual mutant and statistical rules for halting noncompetitive infections will be 
reviewed to determine whether or not to proceed to competitive infections.   
 Immunogenicity or Efficacy Review 
Not applicable 
11.4 Final Analysis Plan 
For both continuous and categorical variables, appropriate summary statistics will be applied.  
For continuous variables, descriptive statistics will include the number of non-missing values, 
mean, standard deviation, median, minimum, and maximum.  For categorical variables, 
descriptive statistics will include counts and percentages per category. 
Baseline and demographic characteristics will be summarized.  All subjects inoculated with a           
dose of N. gonorrhoeae within 1 log
10 of the intended dose and with necessary data elements 
available will be included in the analyses.  Data from subjects who are enrolled in the trial but 
who receive an unintended inoculum dose or who withdraw from the study before objective 
study endpoints are reached will not be included in final analyses.  For noncompetitive 
experimental infections with individual WT or isogenic mutant strains of N. gonorrhoeae, the 
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strain.  To assess the contribution of expression of the gene that was inactivated in the mutant, 
we will use a one-sided Fisher’s Exact Test with significance level of 0.025 to assess the 
difference between the infectivity of WT and mutant strains.   
For mutants that do not demonstrate significantly different infectivity compared to WT, we will 
determine relative fitness of the mutant in competitive infections.  To assess whether the fitness 
of a given mutant is different than that of wild-type, we will compare the ratio of colony-forming 
units of the mutant strain to those of the WT strain at the time of treatment and in the inoculum 
using a Wilcoxon Signed-Rank Test with a significance level of 0.025.   
For noncompetitive infections, multiple analyses are planned to assess the secondary outcome to 
compare the clinical course of infection with mutant and WT N. gonorrhoeae.  We will 
summarize the following by strain: 
• The occurrence of signs and symptoms of urethritis attributable to gonococcal infection by 
day 6  
• The occurrence of bacteriuria (Log10 cfu/mL urine sediment) on day of antibiotic treatment  
• The occurrence of urethritis (> 5.8 Log10 WBC/mL urine sediment) on day of treatment 
• The time from inoculation to treatment 
For noncompetitive infections, analyses of exploratory outcomes will include assessment of host 
responses to infection by summarizing and comparing the following by strain: 
• The detection of local cytokines in urine and systemic cytokines in blood 
• Expression of human genes identified by RNASeq in urine sediment and whole blood from 
infected subjects 
In addition, we will assess bacterial gene expression during experimental infection by comparing 
expression levels of specific gonococcal genes detected by targeted reverse transcriptase PCR 
and at the genome level by bacterial RNASeq from urine from subjects infected with WT vs. 
mutant strains. 


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12 SOURCE DOCUMENTS AND ACCESS TO SOURCE 
DATA/DOCUMENTS 
The investigators at the University of North Carolina at Chapel Hill will maintain appropriate 
research records for this study, in compliance with ICH E6 GCP and regulatory and institutional 
requirements for the protection of human subjects.  As part of conducting this DMID-sponsored 
study, study documentation will be made available to authorized representatives of the sponsor 
and regulatory agencies to examine and when required by law to copy clinical records for the 
purposes of quality assurance reviews, audits, and evaluation of the study safety and progress. 
 
Source documents for this study are maintained in the office of Dr. Marcia Hobbs and in the 
Regulatory Department of the Institute for Global Health and Infectious Diseases.  Documents 
containing identifiable information are kept in locked storage and password-protected electronic 
files.  Source documents include daily trial logs developed by Dr. Hobbs to record daily 
urinalysis and culture results; standard clinic forms for history, physical examinations, clinical 
data and laboratory results.    


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13 QUALITY CONTROL AND QUALITY ASSURANCE 
University of North Carolina research personnel will review study materials produced during this 
study for compliance with the protocol and for accuracy in relation to source documents (Refer 
to Section 12).   All personnel reviewing the study materials will be knowledgeable about the 
study and will have been trained in the ethical and regulatory requirements for protection of 
human research participants in compliance with NIH requirements and The University of North 
Carolina at Chapel Hill “Policy on Education and Certification of Investigators Involved in 
Human Subjects Research.” http://ohre.unc.edu/irbtraining/ 
 
Study records will be reviewed according to the following audit criteria: 
 
1. Meets inclusion criteria (as described in detail in section 5.1) 
2. No exclusion criteria present (as described in detail in section 5.2)   
3. History and physical examination documented 
4. Copies of laboratory reports in chart  
5. Proper informed consent for study participation signed 
6. Copy of informed consents provided to participant 
7. Blood drawing at the time of enrollment does not exceed 45 cc 
8. Correct documentation of daily examinations after inoculation (vital signs and 
examination for urethral discharge) 
9. Treatment with antibiotics documented 
10. Follow-up examination documented and test of cure within 7 days of antibiotic treatment 
11. Follow-up telephone call and any AEs documented   
 
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 for 
clarification and resolution. 


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14 ETHICS/PROTECTION OF HUMAN SUBJECTS 
14.1 Ethical Standard 
The investigator will ensure that this study is conducted in full conformity with the principals set 
forth in the Belmont Report: Ethical Principals and Guidelines for the Protection of Human 
Subjects of Research of the US National Commission for the Protection of Human Subjects of 
Biomedical and Behavioral Research (April 28, 1979) and codified in 45 CFR Part 46 and/or the 
ICH E6; 62 Federal Regulations 25691 (1997). 
14.2 Institutional Review Board 
The investigator will provide for review and approval of this protocol and the associated 
informed consent documents by the University of North Carolina at Chapel Hill Biomedical 
Institutional Review Board (IRB) holding US Federal Wide Assurance #4801 and Office of 
Human Research Protections (OHRP) Registration #s: 00000538, 00000539, 00001648 and 
00001649.  Any amendments to the protocol or consent will be approved prior to their use. 
The principal investigator is responsible for keeping the IRB apprised of the progress of the 
study and of any changes made to the protocol as deemed appropriate, but in any case at least 
once a year. The investigator will also keep the IRB informed of adverse events that affect 
subject safety or protocol violations that affect data integrity. 
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. The 
consent form describing in detail the study interventions/products, study procedures, and risks is 
given to the subject and written documentation of informed consent is required prior to starting 
intervention/administering study product. For this study, written informed consent for all study 
procedures and permission for specimen storage will be obtained both at the screening visit and 
again at enrollment before the study begins.  The consent form 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. At the screening visit, subjects will complete a True/False test of understanding (Appendix 
B) following review of the consent form. The investigator obtaining informed consent will 
review specific sections of the consent form related to questions that the subject does not answer 
correctly.  Following this directed review of the consent form and prior to any procedures being 
done specifically for the study, subjects will sign the informed consent document and initial a 
statement indicating permission for specimen storage.  Subjects who sign the consent form but 
deny permission for specimen storage will be allowed to participate in the trial.  The subjects 
will have the opportunity to discuss the study with their surrogates or think about it prior to 
agreeing to participate. The subjects may withdraw consent at any time throughout the course of 


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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. 
 Informed Consent/Assent Process (in Case of a Minor) 
Not applicable; individuals below the age of 18 will be excluded from the study. 
14.4 Exclusion of Women, Minorities, and Children (Special Populations) 
Females will not be included in the study because of the risk of complications from ascendant 
gonococcal infection.  Male subjects of any race will be included.  Children under 18 years of 
age are deemed inappropriate for inclusion in a study of a sexually transmitted infection. 
14.5 Subject Confidentiality 
Subject confidentiality is strictly held in trust by the participating investigators, their staff, and 
the sponsor(s) and their agents.  Results of study research may be published, but subject 
identities will not be revealed.  After written informed consent is provided, consecutive study 
code numbers will be assigned to the subjects to ensure confidentiality. This study code number 
will be used for laboratory transport and processing, result reporting, and data recording.  
Additionally, the principal investigator will keep records locked in cabinets.  Data entered into 
computerized files will be stored only in a password protected file on a computer in the PI’s 
office, which is accessible only by authorized personnel directly involved with the study. No 
information concerning the study or the data will be released to any unauthorized third party 
without prior written approval of the sponsor. 
This research is covered by a Certificate of Confidentiality from NIH. The researchers with this 
Certificate may not disclose or use information, documents, or biospecimens that may identify 
subjects in any federal, state, or local civil, criminal, administrative, legislative, or other action, 
suit, or proceeding, or be used as evidence, for example, if there is a court subpoena, unless 
subjects have consented for this use. Information, documents, or biospecimens protected by this 
Certificate cannot be disclosed to anyone else who is not connected with the research except, if 
there is a federal, state, or local law that requires disclosure (such as to report child abuse or 
communicable diseases but not for federal, state, or local civil, criminal, administrative, 
legislative, or other proceedings, see below); if subjects have consented to the disclosure, 
including for medical treatment; or if it is used for other scientific research, as allowed by federal 
regulations protecting research subjects.  
The Certificate cannot be used to refuse a request for information from personnel of the US 
federal or state government agency sponsoring the project that is needed for auditing or program 
evaluation by NIAID, which is funding this project, or for information that must be disclosed in 
order to meet the requirements of the US FDA. The study monitor or other authorized 
representatives of the sponsor such as the investigator and her research associates, the UNC-CH 


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Biomedical IRB or its designees, OHRP, the FDA, and DMID 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. The Certificate of Confidentiality does not 
prevent subjects from voluntarily releasing information about themselves or their involvement in 
this research. If subjects want research information released to an insurer, medical care provider, 
or any other person not connected with the research, they must provide consent to allow the 
researchers to release it. 
The Certificate of Confidentiality will not be used to prevent disclosure as required by federal, 
state, or local law of reportable STIs other than experimental gonococcal infection if such 
infections are identified by tests performed for screening or follow up as described in this 
protocol.    
14.6 Study Discontinuation 
In case this study is discontinued, all subjects will receive treatment for termination of infection, 
follow-up examination and test of cure.  
The risk of major complications to subjects in the experimental setting is remote based on the 
natural history of gonorrhea and previous human experimentation.  However, the study will be 
monitored by a SMC to ensure safety and proper performance of the study.  If concerns affecting 
subject safety or data integrity are identified by the SMC, enrollment into the study and study 
procedures will be halted until the identified issues are addressed.    
14.7 Future Use of Stored Specimens 
Bacteria isolated from urine cultures will be stored for future use in studies of gonococcal 
pathogenesis.  Stored urine, serum and isolated peripheral white blood cells from subjects who 
provide permission for stored specimens will be used to measure cytokines, chemokines and 
other molecules that may mediate host immune responses to gonococcal infection.  Specimens 
will be stored in the laboratories of the study investigators on the University of North Carolina 
campus in Chapel Hill, NC.  Stored specimens will be used only for studies approved by the 
University of North Carolina IRB.  All specimens obtained for the purpose of this study become 
the exclusive property of the University of North Carolina at Chapel Hill.  The researchers may 
retain, preserve or dispose of these specimens and may use these specimens for research that may 
result in commercial applications.  Subjects will not be compensated for any future commercial 
use of these specimens. 


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15 DATA HANDLING AND RECORD KEEPING 
The investigator is responsible for ensuring the accuracy, completeness, legibility, and timeliness 
of the data.  All source documents should be neat and in a 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. 
Study visit checklists, logs and flowsheets will be used 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 its designee will provide guidance to investigators on making corrections to the 
source documents and eCRF. 
15.1 Data Management Responsibilities 
All source documents and laboratory reports must be reviewed by the clinical team and verified 
for completeness and accuracy prior to data entry.  Adverse events must be graded, assessed for 
severity and causality, and reviewed by the Principal Investigator or designee. 
Data collection is the responsibility of the clinical trial staff at University of North Carolina at 
Chapel Hill under the supervision of the Principal Investigator. During the study, the investigator 
must maintain complete and accurate documentation. 
The Emmes Corporation will serve as the Statistical and Data Coordinating Center for this study 
and will be responsible for data management, quality review, analysis, and reporting of the study 
data. 
15.2 Data Capture Methods 
Clinical data (including AEs) will be recorded on paper and entered from source documents into 
a 21 CFR Part 11-compliant Internet Data Entry System (IDES) provided by The Emmes 
Corporation.  The data system includes password protection and internal quality checks, such as 
automatic range checks, to identify data that appear inconsistent, incomplete, or inaccurate.   
15.3 Types of Data 
The database will include information under a subject’s coded number about age, race, trial 
dates, inoculum received, whether the subject is infected, whether (and when) he develops signs 
or symptoms of urethritis, and quantitative laboratory data regarding bacteruria and pyuria. 


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15.4 Timing/Reports 
The Emmes Corporation will conduct interim analyses as specified in sections 11.2 and 11.3.  
Results will be included in SMC reports prepared in advance of meetings scheduled as specified 
in section 9.6. 
15.5 Study Records Retention 
Study records will be retained a minimum of 10 years. 
15.6 Protocol Deviations 
A protocol deviation is any noncompliance with the clinical trial protocol, Good Clinical Practice 
(GCP), or protocol–specific 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.  
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 investigator or study staff to use continuous vigilance to identify 
and report deviations within 5 working days of identification of the protocol deviation, or within 
5 working days of the scheduled protocol-required activity.  All deviations must be promptly 
reported to DMID, via The Emmes Corporation’s IDES or via the TRI/ICON DMID-Clinical 
Research Operations and Management Support (CROMS) email 
(protocoldeviations@dmidcroms.com), web- (www.dmidctm.com) or fax-based system (1-215-699-
6288).  As appropriate, corrective actions are to be developed by the site and implemented 
promptly.  Protocol deviations will be maintained in the study binder on a protocol deviation 
form.  Protocol deviations will be reviewed as they occur by the study investigator and study 
coordinator to determine how improvements can be made to prevent future protocol deviations. 
All deviations from the protocol must be addressed in study subject source documents. A 
completed copy of the DMID Protocol Deviation Form (DMID-CROMS form) must be 
maintained in the regulatory file, as well as in the subject’s source document.  Protocol 
deviations must be sent to the local IRB/IEC per their guidelines. The site PI/study staff is 
responsible for knowing and adhering to their IRB/IEC 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 Office of Extramural Research (OER) Grants and Funding, 
http://grants.nih.gov/grants/oer.htm 
 
As of January 2018, all clinical trials supported by the NIH must be registered on 
ClinicalTrials.gov, no later than 21 days after the enrollment of the first subject. Results of all 
clinical trials supported by the NIH, generally, need to be submitted no later than 12 months 
following the primary completion date. A delay of up to 2 years is available for trials that meet 
certain criteria and have applied for certification of delayed posting.  As part of the result posting 
a copy of this protocol (and its amendments) and a copy of the Statistical Analysis Plan will be 
posted on ClincialTrials.gov.  For this trial the responsible party is DMID which will register the 
trial and post results.  The responsible party does not plan to request certification of delayed 
posting. 
 
Refer to: 
•  
Public Law 110-85, Section 801, Clinical Trial Databases 
•  
42CFR11 
• 
NIH NOT-OD-16-149 
 
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. 


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Results of any exploratory analysis will not be published prior to publication of the primary 
results for this study. 
 
*Journal Citation: De Angelis C, Drazen JM, Frizelle FA, Haug C, Hoey J, et al. Clinical trial 
registration: a statement from the International Committee of Medical Journal Editors. N Engl J 
Med. 2004; 351:1250-1 


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17 LITERATURE REFERENCES 
 
1. World Health Organization. Global incidence and prevalence of selected curable sexually 
transmitted infections - 2008. Geneva: WHO Press; 2012. 
2.  Centers for Disease Control and Prevention. Sexually Transmitted Disease Surveillance 
2014. US Department of Health and Human Services. Atlanta, 2015. 
3.  Cohen MS. HIV and sexually transmitted diseases: lethal synergy. Top HIV Med. 
2004;12(4):104-7. 
4.  Cohen MS, Miller WC. Sexually transmitted diseases and human immunodeficiency virus 
infection: cause, effect, or both? Int’l J Infect Dis. 1998;3(1):1-4. 
5.  Cohen MS. Sexually transmitted diseases enhance HIV transmission: no longer a hypothesis. 
Lancet. 1998;351 (suppl III):5-7. 
6.  CDC. Update to CDC’s Sexually Transmitted Diseases Treatment Guidelines, 2010:Oral 
cephalosporins no longer a recommended treatment for gonococcal infections. MMWR. 
2012;61(31):590-4. 
7.  Unemo M, Shafer WM. Antibiotic resistance in Neisseria gonorrhoeae: origin, evolution, 
and lessons learned for the future. Ann N Y Acad Sci. 2011;1230:E19-28. 
8.  Lewis DA. The Gonococcus fights back: is this time a knock out? Sex Transm Infect. 
2010;86(6):415-21. 
9.  Hagman K, Pan W, Spratt BG, Balthazar JT, Judd RC, Shafer WM. Resistance of Neisseria 
gonorrhoeae to antimicrobial hydrophobic agents is modulated by the mtrRCDE efflux 
system. Microbiol. 1995;141:611-22. 
10. Lucas CE, Hagman KE, Levin JC, Stein DC, Shafer WM. Importance of lipooligosaccharide 
structure in determining gonococcal resistance to hydrophobic antimicrobial agents due to 
the mtr efflux system. Mol Microbiol. 1995;16:1001-10. 
11. Shafer WM, Balthazar JT, Hagman K, Morse SA. Missense mutations that alter the DNA-
binding domain of the MtrR protein occur frequently in rectal isolates of Neisseria 
gonorrhoeae that are resistant to faecal lipids. Microbiol. 1995;141:907-11. 
12. Hagman K, Shafer WM. Transcriptional control of the mtr efflux system of Neisseria 
gonorrhoeae. J Bacteriol. 1995;179:4123-8. 
13. Lucas CE, Balthazar JT, Hagman K, Shafer WM. The MtrR repressor binds the DNA 
sequence between the mtrR and mtrC genes of Neisseria gonorrhoeae. J Bacteriol. 
1997;179(13):4123-8. 
14. Jerse AE, Sharma ND, Simms AN, Crow ET, Snyder LA, Shafer WM. A gonococcal efflux 
pump system enhances bacterial survival in a female mouse model of genital tract infection. 
Infect Immun. 2003;71(10):5576-82. 
15. Hobbs MM, Sparling PF, Cohen MS, Shafer WM, Deal CD, Jerse AE. Experimental 
gonococcal infection in male volunteers: Cumulative experience with Neisseria gonorrhoeae 
strains FA1090 and MS11mkC. Front Microbiol. 2011;2:123. 
16. Seifert HS, Wright CJ, Jerse AE, Cohen MS, Cannon JG. Multiple gonococcal pilin antigenic 
variants are produced during experimental human infections. J Clin Invest. 1994;93(6):2744-
9. 


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17. Jerse AE, Cohen MS, Drown PM, Whicker LG, Isbey SF, Seifert HS, Cannon JG. Multiple 
gonococcal opacity proteins are expressed during experimental urethral infection in the male. 
J Exp Med. 1994;179:911-20. 
18. Hobbs MM, Anderson JE, Balthazar JT, Kandler JL, Carlson RW, Ganguly J, Begum AA, 
Duncan JA, Lin JT, Sparling PF, Jerse AE, Shafer WM. Lipid A
's structure mediates 
Neisseria gonorrhoeae fitness during experimental infection of mice and men. MBio. 
2013;4(6):e00892-13. 
19. Anderson JE, Hobbs MM, Biswas GD, Sparling PF. Opposing selective forces for expression 
of the gonococcal lactoferrin receptor. Mol Microbiol. 2003;48(5):1325-37. 
20. Fekety FR. Safety of parenteral third-generation cephalosporins. Am J Med. 
1990;88(4A):38S-44S. 
21. Pichichero ME. Use of selected cephalosporins in penicillin-allergic patients: a paradigm 
shift. Diagn Microbiol Infect Dis. 2007;57(3 Suppl):13S-8S. 
22. Khaliq Y, Zhanel GG. Fluoroquinolone-associated tendinopathy: a critical review of the 
literature. Clin Infect Dis. 2003;36(11):1404-10. 
23. Johnston DM, Cannon JG. Construction of mutant strains of Neisseria gonorrhoeae lacking 
new antibiotic resistance markers using a two gene cassette with positive and negative 
selection. Gene. 1999;236(1):179-84. 
24. Beuzon CR, Holden DW. Use of mixed infection with Salmonella strains to study virulence 
genes and their interactions in vivo. Microbes Infect. 2001;3:1345-52. 
25. Lim EM, Cembrowski G, Cembrowski M, Clarke G.  Race-specific WBC and neutrophil 
count reference intervals.  Int J Lab Hematol. 2010;32:590-7. 
 
 
 


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18 SUPPLEMENTS/APPENDICES 
18.1 Appendix A: Schedule of Events  
 
Evaluation 
Screening 
Visit 
Eligibility Testing  
Entry Visit  Daily follow-up 
for up to 5 days 
Day of 
Antibiotic 
Treatment 
1 week 
follow-up 
visit 
2 week final 
follow-up 
phone call 
Study Day 
-30 to -2 
-2 to -4 
1 
2 to 6 
2 to 6 
5 to 13 
12 to 23 
Study Visit Number 
00A 
00B 
01 
02,03,04,05,06 
02,03,04,05,06 
07 
08 
Review inclusion/ exclusion 
criteria and risks of 
participation; subject signs 
ICF. 
X 
 
X 
 
 
 
 
Subject takes ICF test of 
understanding 
X 
 
 
 
 
 
 
Medical History 
X 
 
 
 
 
 
 
Review Medical History  
 
 
X 
X 
 
X 
 
Review of Medications 
X 
 
X 
X 
 
X 
 
Complete Physical Exam 
X 
 
 
 
 
 
 
Targeted Physical Exam 
 
 
X 
X 
 
X 
 
Vital signs 
X 
 
X 
X 
 
X 
 
Complete Blood Count with 
differential (for WBC, PMN 
and hemoglobin results) 
X 
 
 
 
 
 
 
Serum CH50 testing 
X 
 
 
 
 
 
 
Serum creatinine testing 
X 
 
 
 
 
 
 
Serum ALT testing 
X 
 
 
 
 
 
 


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Evaluation 
Screening 
Visit 
Eligibility Testing  
Entry Visit  Daily follow-up 
for up to 5 days 
Day of 
Antibiotic 
Treatment 
1 week 
follow-up 
visit 
2 week final 
follow-up 
phone call 
Study Day 
-30 to -2 
-2 to -4 
1 
2 to 6 
2 to 6 
5 to 13 
12 to 23 
Study Visit Number 
00A 
00B 
01 
02,03,04,05,06 
02,03,04,05,06 
07 
08 
HIV testing (combination 
Ag/Ab test) 
X 
 
 
 
 
 
 
Syphilis, HBV and HCV 
serologies 
X 
 
 
 
 
 
 
Urine CT/NG and TV 
NAAT  
X 
X 
 
 
 
 
 
Urine CT/NG NAAT (Test 
of cure) 
 
 
 
 
 
X 
 
Urinalysis 
X 
 
 
 
 
X 
 
Intraurethral Inoculation 
 
 
X 
 
 
 
 
Stored blood 
 
X 
 
 
X 
X 
 
Urine culture for       
N. gonorrhoeae 
 
 
 
X 
 
 
 
Urine storage 
 
X 
 
X 
X 
X 
 
Urethral swab  
 
 
 
 
X 
 
 
Antibiotic treatment 
 
 
 
 
X 
 
 
Review Symptoms/Adverse 
Events 
 
 
 
 
 
 
 


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18.2 Appendix B: Subject Comprehension  
These questions will be given to potential study subjects after initial review of informed consent form 
during the screening visit.  Incorrect answers will prompt re-review of the relevant section(s) of consent 
form to clarify the content [indicated in square brackets]. 
 
Instructions:  Respond to the statements below answering T for TRUE or F for FALSE. 
1.  
As a volunteer, I do not have to participate in this research study if I do not want to. [p.1 paragraph 
1; p.7 last paragraph] 
T 
2.  
One goal of this study is to give me a gonorrhea infection with symptoms including urethral 
discharge and discomfort during urination. [p.2 paragraph 2; p.4  paragraph 4] 
T 
3.  
Screening tests to determine if I can participate in this study will not include an HIV test. [p.4 
paragraph 2] 
F 
4.  
The investigators have assured me that there are no risks to me or others associated with my 
participation in this research study. [p.1 paragraph 2; pp.6-7; p.8 paragraph 3]  
F 
5.  
A solution containing live bacteria capable of causing gonorrhea will be delivered through a small 
tube into my penis. [p.4 paragraph 4; p.6 paragraph 2] 
T 
6.  
Gonorrhea is difficult to spread to others and there will be no restrictions on my sexual activity 
during my participation. [p.3 paragraph 2; p.4 paragraphs 1&5; p.5 paragraph 1; p.7 paragraphs 
3&4] 
F 
7.  
As soon as I am discharged from the hospital research unit, I can resume sexual activity without 
risk to my partner(s). [p.3 paragraph 2; p.4 paragraphs 1&5; p.5 paragraph 1; p.7 paragraphs 3&4] 
F 
8.  
The gonorrhea bacteria used to infect me are killed by antibiotics, and all subjects previously 
infected in this research have been cured. [p.6 paragraph 4] 
T 
9.  
Although the bacterial inoculum has been extensively tested for purity, it is still possible that it 
may contain other infectious agents that could infect me. [p.6 paragraph 5] 
T 
10.  
Taking some other medications during the course of this study may influence the safety of the 
experiment. [p.3 paragraph 1] 
T 
11.  
While admitted to the hospital research unit, I will be allowed to leave during the day, but must 
return to give specimens and stay overnight on the ward. [p.4 paragraphs 1&5] 
T 
12.  
The morning urine specimens collected for the study may be from the second or third urination of 
the day. [p.4 paragraph 5; p.5 paragraph 2]  
F 
13.  
Blood and urine specimens that I provide will be stored indefinitely for future research use, unless 
I indicate otherwise. [p.5 paragraph 5;p.9 paragraph 3] 
T 
14.  
If I have any concerns about my participation in this study, I can call the UNC Institutional Review 
Board at 919-966-3113. [p.11 paragraph 1] 
T 


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18.3 Appendix C: Toxicity Table  
Eligibility criteria are based on Grade 1 values in bold below.  
Clinical Adverse Events 
Blood, Serum, or Plasma 
Chemistries  
Reference 
Range1 
Eligibility 
Range2 
L
O/
HI
/N
3 
Mild  
(Grade 
1)4 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Creatinine (mg/dL) 
0.7-1.3 
0.7-1.7 
HI >ULN-1.7 1.8-2.0 
>2.0 
ALT (U/L) 
18 yr 10-40 
10-105 
HI 44-105 
106-175 
>175 
 
19+ yr 19-72 
19-105 
Hematology 
 
 
 
 
 
 
Hemoglobin (Males) (g/dL) 13.5-17.5 12.0-17.5 LO 12.0-12.5 10.0-11.9 <10.0 
White Blood Cell Count 
(WBC) (x10
9/L) 
4.5 – 11.0 2.5-15.0 
HI 11.0-15.0 15.1-20.0 >20.0 
LO 2.50-3.50 1.50-2.49 <1.50 
Neutrophils (ANC) 
(x10
9/L)
5 
2.0-7.5 
1.5-7.5 
LO 1.50-2.00 1.00-1.49 <1.00 
African Americans 1.3-7.5 
1.3-7.5 
 
 
 
 
Urine 
 
 
 
 
 
 
Protein (dipstick) 
Negative 
< 2+ 
HI 1+ 
2+ 
>2+ 
Blood (microscopic) – red 
blood cells per high power 
field (rbc/hpf) 
0-3 
0-10 
HI 5-10 
11-50 
>50 and/or 
gross blood 
1Reference range from UNC Hospitals’ McLendon Clinical Laboratories or reference (25) for ANC for African Americans 
2Laboratory values acceptable for eligibility for enrollment 
3Low, High, Not Graded 
4If initial bound of Grade 1 has gap from reference range or eligibility range, calculations based on NEJM reference ranges 
 
 
 
 
 


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 Urinary frequency 
 Testicular pain 
 Feverish 
 Chills 
 Malaise 
 Diarrhea
 Rash
Objective signs: 
 Urethral discharge  
A first-void urine specimen will be collected.  All subjects will receive antibiotic treatment 
before discharge from the inpatient portion of the study.  Subjects are treated if (1) he requests 
treatment regardless of signs, symptoms or positive cultures (2) purulent urethral discharge is 
observed by the examining clinician or reported by the subject or (3) 5 days after inoculation, 
regardless of infection status.  On the last day of the inpatient portion of the trial, or the day on 
which a subject receives treatment for gonococcal infection, a urethral swab will be obtained.  
Subjects will be observed for approximately 20 minutes following administration of study 
antibiotics. 
Subjects return to the CTRC within one week for re-examination by a physician or physician’s 
assistant licensed to treat gonococcal infections and their complications, and a urine CT/NG 
NAAT test of cure.  The test of cure is a nucleic acid amplification test.  In the unlikely event of 
a positive test of cure, we will obtain a urethral specimen for culture to enable antibiotic 
susceptibility testing.  
8.2 Laboratory Evaluations 
Clinical Laboratory Evaluations 
 Serum CH50 test for complement deficiency, creatinine, ALT and HIV, syphilis, HBV and 
HCV serologies and CBC at screening 
 Urine-based nucleic acid amplification testing for CT/NG and TV and urinalysis at 
screening; urine CT/NG and TV tests 2-4 days prior to enrollment; urine CT/NG test and 
urinalysis at 1 week follow-up (Day 5-13). 
Special Assays or Procedures 
 Daily urine sediment culture for N. gonorrhoeae to monitor infection (Study Days 2-6).  
Bacteria isolated from urine cultures will be enumerated, and individual colonies will be 
stored for research testing to compare numbers and the genotypes and phenotypes of output 
compared to input organisms. 


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 Daily urine dipstick analysis for hematuria (Study Days 2-6). 
 Daily microscopic examination of urine sediment and quantitation of WBCs to monitor 
inflammation (Study Days 2-6).   
 Stored urine (collected 2-4 days prior to enrollment, on Study Days 2-6, and at 1 week 
follow-up [Study Days 5-13]) and blood (collected 2-4 days prior to enrollment, on day of 
antibiotic treatment, and at 1 week follow-up [Study Days 5-13]) from subjects who provide 
permission for stored specimens will be used to measure cytokines, chemokines and other 
molecules that may mediate host immune responses to gonococcal infection.  Levels of 
individual cytokines will be assessed using commercially available ELISAs.  Multiplexed 
cytokine analysis will be measured using slide-based anticytokine antibody arrays or 
microparticle based arrays.  Activation of inflammatory cell death programs will be assessed 
by staining with a fluorescent stain excluded by viable cells followed by quantitation of 
staining by flow cytometry.  Assays of immunological parameters are purely for pursuit of 
research objectives, and results will not be used for clinical purposes. 
Specimen Preparation, Handling, and Shipping 
 Approximately 30 mL of blood will be obtained at screening.  Screening labs will be sent to 
the NC Hospitals McLendon Clinical Laboratories for CH50, CBC, ALT, creatinine and 
HIV, syphilis, HBV and HCV serology tests.  Blood and urine obtained at the eligibility 
testing visit and at followup will be sent to Dr. Hobbs’ Laboratory where specimens will be 
processed and stored at < -70°C according to laboratory SOPs.   
 Urine obtained at screening, before enrollment and at followup will be sent to the NC 
Hospitals McLendon Clinical or UNC CFAR VIM Core Laboratories for nucleic acid 
amplification testing for C. trachomatis, N. gonorrhoeae and T. vaginalis.  Excess urine will 
be transferred to Dr. Hobbs’ Lab, aliquoted and stored frozen at < -70°C. 
 Daily urine specimens will be processed by Dr. Hobbs or other study personnel in the 
investigators’ laboratory at the CTRC.  Urine dipstick analysis will be performed on whole 
urine (8-10 mL).  This volume will then be centrifuged in the investigators’ lab at the CTRC, 
and sediment will be examined microscopically to enumerate WBCs.  The remaining urine 
from the original 50-mL specimen will be centrifuged separately.  The supernatant from the 
spun urine will be aliquoted and stored frozen at < -70°C in Dr. Hobbs’ Lab.  The urine 
sediment will be inoculated onto GCB agar plates for N. gonorrhoeae culture; inoculated 
plates will be loaded into candle jars for transport from the CTRC to Dr. Hobbs’ Lab.  
Individual N. gonorrhoeae colonies isolated from urine cultures (up to 100 per subject per 
day) will be stored frozen at < -70°C in Dr. Hobbs’ Lab. 


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9.2 Methods and Timing for Assessing, Recording, and Analyzing Safety 
Parameters 
Adverse Events  
All AEs that occur from inoculation through the 2 week final follow-up phone call (Study Days 
12-23) will be reported.  At each scheduled visit, AEs will be assessed and any AEs identified 
during this period will be reported.  
The Investigator is responsible for reporting all AEs that are observed or reported during the 
study, regardless of their relationship to study product. 
 
Adverse Event:  ICH E6 Good Clinical Practice Guidelines defines an Adverse Event (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 medicinal (investigational) product.  
The occurrence of an adverse event may come to the attention of study personnel during study 
visits and interviews or by a study recipient presenting for medical care, or upon review by a 
study monitor.    
All AEs including local and systemic reactions not meeting the criteria for “serious adverse 
events” should be captured on the appropriate CRF.  Information to be collected includes event 
description, time of onset, clinician’s assessment of severity, relationship to study product 
(assessed only by those with the training and authority to make a diagnosis, which would include 
MD, PA, Nurse Practitioner, DO, or DDS), and time of resolution/stabilization of the event.  All 
AEs occurring while on study must be documented appropriately regardless of relationship.  All 
AEs will be followed to adequate resolution.  
Any medical condition that is present at the time that the patient is screened should be considered 
as baseline and not reported as an AE.  However, if it deteriorates at any time during the study, it 
should be recorded as an AE. 
All adverse events will be graded for severity and relationship to study product.  
Severity of Event:  All adverse events will be assessed by the investigator according to the 
DMID Toxicity Table (May 2015) as follows: 
  
 Mild: events do not interfere with the patient’s daily activities. 
 Moderate: events may cause some interference with activity not requiring medical 
intervention. 
 Severe: events prevent daily activity and require medical intervention.  


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Changes in the severity of an AE should be documented to allow an assessment of the duration 
of the event at each level of intensity to be performed.  Adverse events characterized as 
intermittent require documentation of onset and duration of each episode. 
Relationship to study products:  The investigator’s assessment of an AE
's relationship to 
study procedure or drug 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.  All adverse events must have their 
relationship to study product assessed using the following 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. 
Relatedness to study product must specify whether to N. gonorrhoeae inoculum, cefixime, 
ceftriaxone or ciprofloxacin. 
Expected AEs are those that occur during or following the study procedures (i.e. urethral 
inoculation, discomfort from collecting specimens, bruising from blood draw) or study 
medication administration which are outlined in the potential risks of the study medication 
package inserts and have no other definitely related explanation. Unexpected AEs are those 
events that occur and are not listed in the package insert for the study medication or events that 
are not the usual side effects associated with the study procedures. 
Reactogenicity (for Vaccine Studies and Some Therapeutic Trials) 
Not applicable 
Serious Adverse Events 
Suspected adverse reaction means any adverse event for which there is a reasonable possibility 
that the drug caused the adverse event.  For the purposes of IND safety reporting, “reasonable 
possibility” means there is evidence to suggest a causal relationship between the drug and the 
adverse event.  Suspected adverse reaction implies a lesser degree of certainty about causality 
than adverse reaction, which means any adverse event caused by a drug. 
Unexpected adverse event or unexpected suspected adverse reaction.  An adverse event or 
suspected adverse reaction is considered “unexpected” if it is not listed in the investigator 


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Procedures to be Followed in the Event of Abnormal Laboratory Test Values or 
Abnormal Clinical Findings 
Abnormal urine dipstick values will be reported to the study physician who will follow through 
with appropriate clinical testing, care and treatment. 
9.3 Reporting Procedures 
Serious Adverse Events 
Adverse Events 
Adverse Events including local and systemic reactions not meeting the criteria for “serious 
adverse events” will be captured in the investigator’s records for the subject.  Information to be 
collected includes event description, time of onset, investigator assessment of severity, 
relationship to study product, time of resolution of the event, seriousness, and outcome.  All 
adverse events occurring while on study must be documented appropriately regardless of 
relationship. All AEs will be followed to adequate resolution.  
Any medical condition that is present at the time that the patient is screened should be considered 
as baseline and not reported as an AE.  However, if it deteriorates at any time during the study it 
should be recorded as an AE. 
Serious Adverse Events 
All serious adverse events will be:
 recorded on the appropriate serious adverse event case report form 
 followed through resolution by a study physician 
 reviewed by a study physician 
Regulatory Reporting for Studies Conducted Under DMID-Sponsored IND 
All SAEs will be submitted 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 


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Other supporting documentation of the event may be requested by the DMID Pharmacovigilance 
Group and should be provided as soon as possible. 
The DMID medical monitor and clinical protocol 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 investigator becomes aware of an SAE that is 
suspected to be related to study product, the investigator will report the event to the DMID 
Pharmacovigilance Group. 
Regulatory Reporting for Studies Not Conducted Under DMID-Sponsored IND 
Not applicable 
Other Adverse Events (if applicable) 
The occurrence of transmission of N. gonorrhoeae to a non-study subject will be reported.  This 
has not occurred in prior studies.  If an enrolled subject were to report having had sex during the 
timeframe relevant to study activities, the partner(s) would be referred to the UNC infectious 
diseases clinic, evaluated and given empiric treatment.  Specimens would be obtained from the 
partner(s) to determine whether the infecting organism matched the inoculated subject’s 
infection. The partner would be asked to provide informed consent to undergo non-standard of 
care procedures to evaluate the infection.  
The occurrence of N. gonorrhoeae inoculum contamination and, if applicable, clinical events 
resulting from contamination, microbiological failure of cefixime or ceftriaxone treatment post 
infection, and response to ciprofloxacin in treatment failures will be reported to DMID 
Pharmacovigilence to be forwarded to DMID and the SMC and to the on-site ISM within 2 
weeks of occurrence.  If these events meet criteria for serious adverse event, they will be 
reported on an SAE form according to SAE reporting procedures. 
 Site SAEs will be reported to DMID Pharmacovigilance according to the protocol and, 
separately, to the on-site ISM.  DMID Pharmacovigilance will forward SAEs to DMID and, 
separately, to the DMID SMC Coordination staff for forwarding to the SMC. 
 Any serious and unexpected (not listed in the package insert) adverse events considered to be 
related to licensed products that are administered in the course of the study (cefixime, 
ceftriaxone, ciprofloxacin, lidocaine, epinephrine) will be reported via DMID 
Pharmacovigilance to the FDA and separately to the on-site ISM. DMID Pharmacovigilance 
will forward SAEs to DMID and, separately, to the DMID SMC Coordination staff for 
forwarding to the SMC. 


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Reporting of Pregnancy 
Not applicable 
9.4 Type and Duration of Follow-up of Subjects after Adverse Events 
Subjects who experience an adverse event will be followed until symptoms resolve or stabilize.  
Follow-up will continue through the duration of scheduled study visits.  If a subject has 
completed the required study visits and symptoms persist, the subject will be monitored by 
telephone or clinic visit until the adverse event resolves or stabilizes.  
9.5 Halting Rules 
The risk of major complications to subjects in the experimental setting is remote based on the 
natural history of gonorrhea and previous human experimentation.  However, the study will be 
monitored by a Safety Monitoring Committee (SMC) to ensure safety and proper performance of 
the study.  If concerns affecting subject safety or data integrity are identified by the SMC, no 
new subjects will be enrolled, but protocol procedures will continue, to ensure the safety of the 
subjects who have been inoculated.  The trial will be halted (at least temporarily) if at any time 
during the study: 
1. One SAE related to study procedure or study product(s) 
2. Three or more severe AEs of the same type (described by the same MedDRA 
SOC and Preferred Term) related to study procedure or study product(s) 
3. One antibiotic treatment failure (so that susceptibility of the isolate may be 
evaluated) 
4. Documented contamination of inoculum 
5. A late, or systemic complication of gonococcal infection including any of the 
following is reported: 
a. recovery of the infecting N. gonorrhoeae strain(s) from a normally sterile 
extragenital site 
b. petechial or pustular acral skin lesions  
c. asymmetric polyarthralgia  
d. tenosynovitis  
e. oligoarticular septic arthritis 


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For competitive infections with mixed inocula, sample sizes are determined by the expected 
standard deviation of the mean proportion of recovered WT gonococci.  Because calculations are 
based on the numbers of bacteria, constituting larger sample sizes than infectious dose 
calculations based on the number of infected subjects, mixed infections can provide a more 
accurate indication of attenuation using fewer subjects than traditional comparisons of infectivity 
using separate, inocula with individual strains (24).  Note that competitive infections will not 
necessarily be conducted for isogenic mutants that demonstrate statistically significant 
differences in the primary and secondary outcomes specified for noncompetitive infections.  The 
total number of subjects specified in the current protocol (n = 32) refers to the maximum number 
of participants to be prospectively enrolled in noncompetitive and competitive studies for the 
MtrD mutant.  The total of 32 prospectively enrolled subjects for the combined MtrD mutant 
studies provides sufficient sample sizes for both noncompetitive and competitive analyses as 
described. 
11.3 Planned Interim Analyses (if applicable) 
Infectivity Outcomes Review 
Interim analyses for noncompetitive infection studies will include results from up to 8 evaluable 
subjects inoculated with the WT N. gonorrhoeae strain and at least 4 evaluable subjects 
inoculated with the isogenic mutant (See Table 2 above). For interim analyses of noncompetitive 
infections, data for WT may include results from the current working bank, regardless of the 
proximity in time of the specific isogenic mutant group. See Section 5.3.4 for handling of 
withdrawals.  
At the interim analysis, the following decision rule for the noncompetitive analysis will be used: 
 If the p-value from Fisher’s Exact Test is less than 0.025, we will consider stopping the 
mutant group.  
 If the p-value from Fisher’s Exact Test is greater than 0.025 and the p-value will still be 
greater than 0.025 regardless of the results from additional subjects in the mutant group, we 
may consider closing enrollment for the noncompetitive infection study early for futility and 
proceeding with the competitive infection study. 
 If the p-value from Fisher’s Exact Test is greater than or equal to 0.025, we will continue to 
enroll until evaluable data are obtained from at least 7 subjects in the mutant group.     
If additional subjects are enrolled in the mutant group, a second interim analysis will include 
results from all evaluable subjects in the mutant and WT groups.  At the second interim analysis, 
the following decision rule for the noncompetitive analysis will be used: 
 If the p-value from Fisher’s Exact Test is less than 0.025, we will consider stopping studies 
for the isogenic mutant.  


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 If the p-value from Fisher’s Exact Test is greater than or equal to 0.025, we will conclude that 
a difference in infectivity cannot be determined in noncompetitive experimental human 
infections.     
 
If the infectivity of the two strains is not significantly different at that point, we will proceed with 
competitive infections to determine the relative fitness of the isogenic mutant. Interim analyses 
for competitive infections will be conducted after at least 5 subjects have been inoculated with a 
specific mixture of WT and mutant.  The following decision rule will be used for the interim 
analysis: 
 If the p-value from the sign test of the competitive index is less than 0.025, we will consider 
stopping the competitive infection group 
 If the p-value from the sign test of the competitive index is greater than or equal to 0.025, we 
will continue to enroll up to 16 subjects. 
Safety Review  
The SMC will convene to review subject safety as described in section 9.6.  Safety outcome 
measures include adverse events, protocol deviations and any unexpected events that occurred 
since the previous review.  Primary and secondary study outcomes for subject groups enrolled to 
test an individual mutant and statistical rules for halting noncompetitive infections will be 
reviewed to determine whether or not to proceed to competitive infections.   
Immunogenicity or Efficacy Review 
Not applicable 
11.4 Final Analysis Plan 
For both continuous and categorical variables, appropriate summary statistics will be applied.  
For continuous variables, descriptive statistics will include the number of non-missing values, 
mean, standard deviation, median, minimum, and maximum.  For categorical variables, 
descriptive statistics will include counts and percentages per category. 
Baseline and demographic characteristics will be summarized.  All subjects inoculated with a           
dose of N. gonorrhoeae within 1 log
10 of the intended dose and with necessary data elements 
available will be included in the analyses.  Data from subjects who are enrolled in the trial but 
who receive an unintended inoculum dose or who withdraw from the study before objective 
study endpoints are reached will not be included in final analyses.  For noncompetitive 
experimental infections with individual WT or isogenic mutant strains of N. gonorrhoeae, the 
primary outcome is the proportion of subjects that become infected after inoculation with each 


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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. 
Informed Consent/Assent Process (in Case of a Minor)
Not applicable; individuals below the age of 18 will be excluded from the study. 
14.4 Exclusion of Women, Minorities, and Children (Special Populations) 
Females will not be included in the study because of the risk of complications from ascendant 
gonococcal infection.  Male subjects of any race will be included.  Children under 18 years of 
age are deemed inappropriate for inclusion in a study of a sexually transmitted infection. 
14.5 Subject Confidentiality 
Subject confidentiality is strictly held in trust by the participating investigators, their staff, and 
the sponsor(s) and their agents.  Results of study research may be published, but subject 
identities will not be revealed.  After written informed consent is provided, consecutive study 
code numbers will be assigned to the subjects to ensure confidentiality. This study code number 
will be used for laboratory transport and processing, result reporting, and data recording.  
Additionally, the principal investigator will keep records locked in cabinets.  Data entered into 
computerized files will be stored only in a password protected file on a computer in the PI’s 
office, which is accessible only by authorized personnel directly involved with the study. No 
information concerning the study or the data will be released to any unauthorized third party 
without prior written approval of the sponsor. 
This research is covered by a Certificate of Confidentiality from NIH. The researchers with this 
Certificate may not disclose or use information, documents, or biospecimens that may identify 
subjects in any federal, state, or local civil, criminal, administrative, legislative, or other action, 
suit, or proceeding, or be used as evidence, for example, if there is a court subpoena, unless 
subjects have consented for this use. Information, documents, or biospecimens protected by this 
Certificate cannot be disclosed to anyone else who is not connected with the research except, if 
there is a federal, state, or local law that requires disclosure (such as to report child abuse or 
communicable diseases but not for federal, state, or local civil, criminal, administrative, 
legislative, or other proceedings, see below); if subjects have consented to the disclosure, 
including for medical treatment; or if it is used for other scientific research, as allowed by federal 
regulations protecting research subjects.  
The Certificate cannot be used to refuse a request for information from personnel of the US 
federal or state government agency sponsoring the project that is needed for auditing or program 
evaluation by NIAID, which is funding this project, or for information that must be disclosed in 
order to meet the requirements of the US FDA. The study monitor or other authorized 
representatives of the sponsor such as the investigator and her research associates, the UNC-CH