Device Trial: Identification of Herpes Simplex Virus (HSV) Shedding in the Female Genital Tract of Pregnant and Nonpregnant Women by GeneXpert PCR, Routine PCR, and Culture
Conditions: Herpes Simplex Virus
Sponsor: University of Alabama at Birmingham

 
 
 
 
 
IDENTIFICATION OF HERPES SIMPLEX VIRUS (HSV) SHEDDING IN 
THE FEMALE GENITAL TRACT OF PREGNANT AND NONPREGNANT 
WOMEN BY THE XPERT HSV 1/2 ASSAY, ROUTINE PCR, AND 
CULTURE 
DMID Protocol Number:  11-0070 
 
DMID Funding Mechanism:  HHSN272201100034C 
Industry Support Provided by:  Cepheid 
Other Identifying Numbers:  Cepheid No. 174 
Sponsor:  DMID 
Principal Investigator:  David W. Kimberlin, M.D. 
DMID Clinical Project Manager:  Walla Dempsey, Ph.D. 
Draft or Version Number:  4.0 
 3 July 2018 
 
 
 

Collaborative Antiviral Study Group 
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STATEMENT OF COMPLIANCE 
Each investigator must adhere to the protocol as detailed in this document.  Each 
investigator will be responsible for enrolling only those study participants who have met 
protocol eligibility criteria.  This trial will be conducted in compliance with the protocol, 
International Conference on Harmonization Good Clinical Practice E6 (ICH-GCP), and 
the applicable regulatory requirements, including: 
• U.S. Code of Federal Regulations applicable to clinical studies (45 CFR 46 and 21 
CFR including parts 50 and 56 concerning informed consent and IRB regulations, 
and part 812 subparts C, G and E). 
• Completion of Human Subjects Protection Training.  Refer to 
http://grants.nih.gov/grants/guide/notice-files/NOT-OD-01-061.html; 
http://cme.cancer.gov/clinicaltrials/learning/humanparticipant-protections.asp  

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PROTOCOL SIGNATURE PAGE 
The signature below constitutes the approval of this protocol “Identification of Herpes 
Simplex Virus (HSV) Shedding in the Female Genital Tract of Pregnant 
and Nonpregnant Women by the Xpert HSV 1/2 Assay, Routine PCR, 
and Culture” and the attachments, and provides the necessary assurances that this trial will 
be conducted according to all stipulations of the protocol, including all statements regarding 
confidentiality, and according to local legal and regulatory requirements and applicable U.S. 
federal regulations and ICH guidelines.  It is understood that no deviations from the protocol 
may be made without permission of the Sponsor. 
 
Site Investigator: 
 
Signed: 
 
Date: 
 
Name 
 
 
 
                Title 

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TABLE OF CONTENTS 
Page 
Statement of Compliance……………………………………………………………………………….. ii 
Protocol Signature Page………………………………………………………………………………… iii 
Table of Contents…………………………………………………………………………………………iv 
List of Abbreviations…………………………………………………………………………………….. vi 
Protocol Summary………………………………………………………………………………………. vii 
1 
Key Roles……………………………………………………………………………………………1 
2 
Background Information and Scientific Rationale……………………………………………… 6 
2.1 
Background Information .......................................................................................... 6 
2.2 
Scientific Rationale ................................................................................................. 8 
2.3 
Potential Risks and Benefits ................................................................................... 9 
2.3.1 
Potential Risks ............................................................................................ 9 
2.3.2 
Known Potential Benefits............................................................................. 9 
3 
Objectives………………………………………………………………………………………….10 
3.1 
Study Objectives ................................................................................................... 10 
3.1.1 
Primary Objectives .................................................................................... 10 
3.1.2 
Secondary Objectives ............................................................................... 10 
3.1.3 
Exploratory Objectives .............................................................................. 10 
3.2 
Study Outcome Measures .................................................................................... 11 
3.2.1 
Primary Outcome Measures ...................................................................... 11 
3.2.2 
Secondary Outcome Measures ................................................................. 11 
3.2.3 
Exploratory Outcome Measures ................................................................ 11 
4 
Study Design………………………………………………………………………………………12 
4.1 
Substudies (if applicable) ...................................................................................... 13 
5 
Study Population…………………………………………………………………………………. 14 
5.1 
Recruitment Strategies ......................................................................................... 14 
5.2 
Subject Inclusion Criteria ...................................................................................... 14 
5.3 
Subject Exclusion Criteria ..................................................................................... 15 
6 
Study Procedures/Evaluations…………………………………………………………………..16 
6.1 
Test Device ........................................................................................................... 16 
6.2 
Study Procedures ................................................................................................. 16 
6.3 
Laboratory Evaluations ......................................................................................... 18 
6.3.1 
Laboratory Evaluations/Assays ................................................................. 18 
6.3.2 
Specimen Preparation, Handling, and Shipping ........................................ 19 
7 
Statistical Considerations……………………………………………………………………… 20 
7.1 
Study Hypotheses ................................................................................................. 20 
7.2 
Sample Size Considerations ................................................................................. 20 
7.3 
Participant Enrollment and Follow-Up ................................................................... 22 
7.4 
Analysis Plan ........................................................................................................ 23 

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8 
Source Documents and Access to source data/documents………………………………… 24 
9 
Quality Control and Quality Assurance…………………………………………………………25 
10 
Subject Confidentiality…………………………………………………………………………… 26 
10.1 
Future Use of Stored Specimens .......................................................................... 26 
11 
Informed Consent Process……………………………………………………………………… 28 
11.1 
Informed Consent/Assent Process (in Case of a Minor or Others Unable to 
Consent for Themselves) ...................................................................................... 29 
11.2 
Institutional Review Board .................................................................................... 29 
12 
Clinical Monitoring Structure……………………………………………………………………. 31 
12.1 
Site Monitoring Plan .............................................................................................. 31 
13 
Data Handling and Record Keeping…………………………………………………………… 32 
14 
Publication Policy………………………………………………………………………………… 35 
15 
Literature References……………………………………………………………………………. 36 
Supplements/Appendices……………………………………………………………………………… 39 
Appendix A: Schedule of Events……………………………………………………………………… 40 
 
 

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LIST OF ABBREVIATIONS 
AE 
Adverse Event/Adverse Experience 
CASG 
Collaborative Antiviral Study Group 
CFR 
Code of Federal Regulations 
DHHS 
Department of Health and Human Services 
DMID 
Division of Microbiology and Infectious Diseases, NIAID, NIH, 
DHHS 
DNA 
Deoxyribonucleic Acid 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
HIV 
Human Immunodeficiency Virus 
HSV 
Herpes Simplex Virus 
ICF 
Informed Consent Form 
ICH 
International Conference on Harmonisation 
IDE 
Investigational Device Exemption 
IEC 
Independent or Institutional Ethics Committee 
IND 
Investigational New Drug Application 
IRB 
Institutional Review Board 
MOP 
Manual of Procedures 
N 
Number (typically refers to subjects) 
N/A 
Not Applicable 
NIAID 
National Institute of Allergy and Infectious Diseases, NIH, DHHS 
NIH 
National Institutes of Health 
OHRP 
Office for Human Research Protections 
OHSR 
Office for Human Subjects Research 
PCC 
Probe Check Control 
PCR 
Polymerase Chain Reaction 
PI 
Principal Investigator 
SAC 
Sample Adequacy Control 
SPC 
Sample Processing Control 
STI 
Sexually Transmitted Infections 
UAB 
University of Alabama at Birmingham 
US 
United States 

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PROTOCOL SUMMARY 
Title: 
Identification of Herpes Simplex Virus (HSV) Shedding in the 
Female Genital Tract of Pregnant and Nonpregnant Women by 
the Xpert HSV 1/2 Assay, Routine PCR, and Culture 
Phase: 
N/A (Device Trial) 
Population: 
Group 1:  nonpregnant women in Sexually Transmitted 
Infections (STI) Clinics with clinically-apparent HSV lesions; and 
Group 2:  pregnant women admitted with the intent of delivery 
with no visible evidence of HSV infection 
Number of Sites: 
15 (listed in Section 1) 
Study Duration: 
5 years 
Subject Participation 
Duration: 
1 day for Group 1 (nonpregnant women); 30-90 days for Group 
2 (asymptomatic pregnant women) 
Objectives: 
 
 
Primary: 
• To estimate the sensitivity of the Xpert HSV 1/2 Assay 
relative to culture for detecting herpes simplex virus (HSV) 
DNA in the genital tract of nonpregnant women in sexually 
transmitted infections (STI) clinics 
• To estimate the positive percent agreement and negative 
percent agreement  of the Xpert HSV 1/2 Assay relative to 
routine PCR for detecting HSV DNA in the genital tract of 
pregnant women admitted with the intent of delivery 
Secondary: 
• To estimate the positive predictive value of the Xpert HSV 
1/2 Assay relative to culture for detecting HSV DNA in 
genital tract of nonpregnant women in STI clinics  
• To estimate the positive percent agreement of the Xpert 
HSV 1/2 Assay relative to routine  PCR  for detecting HSV 
DNA in genital tract of nonpregnant women in STI clinics 
• To compare the performance of Xpert with routine PCR in 
the combined group (pregnant and non-pregnant women) in 
detecting HSV-1 and HSV-2 virus, separately 
• To quantify HSV viral load, as determined by routine PCR, 
in the genital tract of women shedding the virus 
• To assess the type of infection (first-episode primary, first-
episode non-primary, recurrent) among women shedding 

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HSV 
Exploratory: 
• To determine rates of neonatal HSV disease among 
neonates delivered to pregnant women without active 
genital HSV lesions 
• To assess likelihood of viral transmission to neonates 
exposed to HSV during delivery as a manifestation of 
mode of delivery 
Description of Study 
Design: 
The natural history of genital HSV shedding from the genital 
tract of pregnant and non-pregnant women will be evaluated, 
and the utility of a new cartridge-based PCR platform (the 
GeneXpert system with the Xpert HSV 1/2 Assay cartridge) for 
detection of HSV-1 or HSV-2 will be assessed.  Two groups of 
women will be studied:  1) Group 1 consists of non-pregnant 
women who present at an STI clinic (likely at one or two study 
centers) with clinically evident HSV lesions; and 2) Group 2 
consists of pregnant women presenting with the intent to deliver 
with no evidence of HSV lesions (at all study centers). 
For Group 1 (n=300), viral shedding will be assessed by viral 
culture in real time, followed by detection of HSV-1 and HSV-2 
DNA by two PCR methods (routine PCR and the Xpert HSV 1/2 
PCR using the GeneXpert platform).  PCR detection will be 
completed in batches and not in real time.  Results from the 
GeneXpert system (Xpert HSV 1/2 PCR) will be compared 
against results from HSV viral cultures and routine PCR. 
For Group 2 (n=12,500), viral shedding will be assessed by PCR 
methodology only (routine PCR and Xpert HSV 1/2 PCR).  
Approximately half of the women in Group 2 will be tested by 
routine HSV PCR and Xpert HSV 1/2 PCR; specimens from the 
rest of the women in Group 2 will be stored for possible testing 
in the future by routine HSV PCR and Xpert HSV 1/2 PCR.  In 
this manner, we will maximize the data from which to compare 
Xpert HSV 1/2 PCR results with routine PCR, while maintaining 
flexibility to ensure an adequate number of specimens are 
positive for HSV DNA by routine PCR. 
All Xpert and routine PCR testing  will be performed at the UAB 
Central Laboratory. 
A blood specimen for determination of HSV type-specific 

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antibodies will be obtained on Study Day 1 from all women in 
both groups (Group 1 and Group 2).  If she is determined to be 
shedding HSV in the genital tract (using any of the three viral 
methods), then type-specific serologic testing will be performed.  
Correlation of viral typing from the virologic sampling with HSV-1 
and HSV-2 serostatus will allow for categorization of infection 
(first-episode primary, first-episode nonprimary, or recurrent 
infection). 
HSV culture results from women in Group 1 will be reported to 
their treating physician.  Virologic results from women in Group 
2 will not be reported back to the subject or her physician.  The 
reasons for not reporting results to the Group 2 subject or her 
physician are two-fold:  1) the PCR analyses are performed in 
batches, so no results will be available within a timeframe in 
which clinical decisions on the woman’s care could be made; 
and 2) routine HSV PCR and Xpert HSV 1/2 Assay are not FDA-
cleared tests, so at this point the reliability of a positive or a 
negative test result is not known.  Even if PCR results (routine 
HSV PCR or Xpert HSV 1/2 Assay) could be obtained in a real-
time (non-batched) fashion, it would be irresponsible to report 
results from a non-validated test to the subject and her health 
care provider, as they may be influenced to make clinical 
decisions based on this unvalidated information. Furthermore, 
using the results of this research test for clinical decision making 
is not the intent of this study protocol.  All pregnant women in 
Group 2 will receive written materials on Study Day 1 educating 
them on signs and symptoms of neonatal HSV disease.  All 
postpartum women or designated representative will be 
contacted 30-90 days post-delivery and an inquiry will be made 
to determine mode of delivery and whether their babies 
developed neonatal HSV disease. 
Data on the incidence of neonatal HSV disease among babies 
delivered to women in Group 2 will be compared with the 
incidence data from Brown et al.1  In their study of almost 
60,000 women conducted over a 20 year period, this group of 
researchers has reported an incidence rate for neonatal HSV 
disease of 1 in 3,200 live births. 
Estimated Time to 
Complete Enrollment: 
4 years from enrollment of the first study subject 

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*Schematic of Study Design: 
 
 
 
 
 
 
 
 
 
 
 
 
Nonpregnant women with symptomatic genital 
HSV infection 
Pregnant women with no clinical evidence of 
genital HSV infection (asymptomatic) 
N=300                                      
(Group 1) 
N=12,500 
(Group 2) 
Genital swab for HSV culture, routine HSV PCR, 
and Xpert HSV 1/2 PCR; serology for HSV if 
PCR- or culture-positive 
Genital swab; serology for HSV if PCR-positive 
• 
Approximately half will be evaluated by 
routine HSV PCR and Xpert HSV PCR; 
serology for HSV if PCR-positive 
• 
The other half will be banked for 
possible future virologic testing 
Study population 
Informed Consent and 
Enrollment 
Study Day 1 
Procedure 
Women in Group 2 will be contacted to collect 
information on the method of delivery and 
whether their neonate(s) had evidence of HSV 
disease 
Study Day 30-90 
Follow-up 

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1 
KEY ROLES 
Individuals: 
DMID Clinical Project Manager: 
Walla Dempsey, PhD 
 Division of Virology 
DMID/NIAID/NIH 
Division of Microbiology and Infectious Diseases, NIAID, NIH  
Room 8E57 
5601 Fishers Lane MSC 9825  
Bethesda, MD  20892-9825 (Zip for express mail use Rockville, 
MD 20852 
Phone: 240-292-4197 
FAX: 301-480-1594 
wdempsey@niaid.nih.gov 
 
Principal Investigator: 
David W. Kimberlin, M.D. 
Protocol Chair 
Principal Investigator, Collaborative Antiviral Study Group 
Professor of Pediatrics 
Sergio Stagno Endowed Chair in Pediatric Infectious Diseases 
The University of Alabama at Birmingham 
1600 Seventh Avenue South, CHB 303 
Birmingham, AL  35233 
Phone: 205-934-5316 
Phone: 205-996-6097 (direct line) 
FAX: 205-975-9972 
e-mail: dkimberlin@peds.uab.edu 
 
Clinical Studies Administrator 
Ilet Dale, RN, BSN, MSM, CCRP 
Collaborative Antiviral Study Group 
The University of Alabama at Birmingham 
1600 Seventh Avenue South, CHB 303 
Birmingham, AL  35233 
Phone: 205-934-5316 
Phone: 205-996-7809 (direct line) 
FAX: 205-934-8559 
e-mail: idale@peds.uab.edu 
 
DMID Medical Officer: 
Jill E. Long, MD/MPH, FACOG 
Department of Health and Human Services 

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National Institutes of Health 
5601 Fishers Lane, Room 8E45 
Rockville Maryland 20892-9825  
Phone: 240-627-3305 
Fax:     (301) 480-3617 
E-mail: jill.long@nih.gov 
 
Institutions: 
 
University of Alabama at Birmingham  
David W. Kimberlin, M.D. 
 
Department of Pediatrics, Division of Infectious Diseases  
1600 7th Avenue South, CHB 303 
 
Birmingham, AL 35233 
 
Phone: 205-934-5316  
Fax: 205-975-9972 
 
Email: Dkimberlin@peds.uab.edu  
 
University of Arkansas for Medical Sciences  
Jose Romero, MD 
 
Arkansas Children's Hospital  
800 Marshall Street 
 
Pediatric Infectious Diseases, Slot 512-11 
 
Little Rock, AR 72202  
Phone: 501-364-1416  
Fax: 501-364-3551 
 
Email: RomeroJose@uams.edu 
 
University of Colorado at Denver Health Sciences Center  
Mark J. Abzug, M.D. 
University of Colorado Denver 
 
The Children's Hospital - Pediatric Infectious Disease 
 
13123 East 16th Avenue   Box B055  
Aurora, CO 80045 
 
Phone: 720-777-6389  
Fax: 720 777 7295 
 
Email: : mark.abzug@childrenscolorado.org  
 
Emory Children's Center  
Andrea (Andi) Shane, M.D., M.P.H.  
2015 Uppergate Drive NE 
 
Rm. 534 
 
CELL NUMBER:  404-354-7692 
 
Atlanta, GA 30322 
 
Phone: 404-727-9880  
Fax: 404-727-9223 
 
Email: andi_shane@oz.ped.emory.edu 
 

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Louisiana State University Health Science Center – Shreveport 
John Vanchiere, MD, PhD 
1501 Kings Highway, #5-303 
Shreveport, LA  71103 
Phone: 318-675-7877  
Fax: 318-675-6059 
 
Email: jvanch@lsuhsc.edu 
 
Washington University in St Louis School of Medicine  
Gregory Storch, M.D.  
School of Med. at St. Louis Children's Hospital 
 
660 S. Euclid  
Campus Box 8116 
 
St. Louis, MO 63110  
Phone: 314-454-6079  
Fax: 314-747-1404 
 
Email: storch_G@kids.wustl.edu 
 
Dartmouth Medical School 
Peter F. Wright, M.D. 
Professor of Pediatrics 
Division of Infectious Disease and International Health 
1 Medical Center Drive, 
Lebanon, New Hampshire 03756 
Tel- 603-653-6190 
e-mail- peter.f.wright @dartmouth.edu 
 
University of Rochester Medical Center 
 
Mary Caserta, M.D. 
 
601 Elmwood Avenue 
Box 690 
 
 
Rochester, NY 14642  
Phone: 585-275-5944  
Fax: 585-273-1104 
 
Email: mary_caserta@urmc.rochester.edu 
 
Steven & Alexandra Cohen Children’s Medical Center of New 
York (CCMC) 
Sunil Sood, MD 
 
300 Community Drive  
9 Tower North Shore University Hospital, Room 8  
 
Manhasset, NY 11030 
 
Phone: 516-562-3957  
Fax: 516-562-3958 
 
Email: sood@lij.edu 
 
Carolinas Medical Center - Charlotte  
Amina Ahmed, M.D. B.S. 
 

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Department of Pediatrics 
 
1000 Blythe Boulevard 
MEB-4th 
 
Charlotte, NC 28203  
Phone: 704-381-6870  
Fax:   
Email: Amina.ahmed@carolinashealthcare.org 
 
MetroHealth Medical Center 
Nazha F. Abughali, M.D. 
 
Department of Pediatrics 
2500 MetroHealth Drive 
Old Research Bldg  Rm 350 
Cleveland, OH 44109 
Phone: 216-778-3402 
Fax: 216-778-2435 
Email: nabughali@metrohealth.org 
 
McGee Women’s Hospital 
Harold Wiesenfeld MD 
Children's Hospital of Pittsburgh of UPMC 
 
Marian G. Michaels, M.D., M.P.H. 
One Children's Hospital Drive 
4401 Pen Avenue, AOB 3rd Floor 
 
Pittsburgh, PA 15224  
Phone: 412-692-6786/4129158191  
Fax: 412-864-8977 
 
Email: marian.michaels@chp.edu 
 
Nationwide Children’s Hospital 
Pablo Sanchez, MD  
Ohio State University  
The Research Institute at Nationwide Children’s Hospital 
 
700 Children’s Drive, RB3, WB5215 
Columbus Ohio 43205-2664 
Phone: 614-355-5724 
Fax: 614-355-5899 
 
Email: Pablo.Sanchez@nationwidechildrens.org 
 
University of Utah School of Medicine 
Kwabena Ampofo, MD 
30 North 1900 East  
SOM Room # 2A100 
Salt Lake City, UT  84132 
Phone: 801-581-6791 
Fax: 801-585-3789 
Email: krow.ampofo@hsc.utah.edu 
 
Children's National Medical Center  

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Roberta L. DeBiasi, MD 
 
111 Michigan Avenue, NW 
 
Pediatric Infectious Diseases, West Wing 3.5 
 
Washington, DC 20010 
 
Phone: 202-476-5051  
Fax: 202-476-3850 
 
Email: rdebiasi@cnmc.org 
Optional: 
 
Statistician: 
Inmaculada "Chichi" Aban, PhD 
Associate Professor 
Department of Biostatistics 
School of Public Health 
The University of Alabama at Birmingham 
RPHB 327 
Phone: (205) 934-2732 
Fax: 
(205) 975-2541 
e-mail: CAban@ms.soph.uab.edu 
 
Industry Collaborator: 
David H. Persing, M.D., Ph.D. 
Executive Vice President 
Chief Medical & Technology Officer 
Cepheid Corporation 
904 Caribbean Drive 
Sunnyvale, CA  94089 
Phone: 408-400-8207 
Fax: 
408-541-6439 
e-mail: david.persing@cepheid.com 
 

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2 
BACKGROUND INFORMATION AND SCIENTIFIC 
RATIONALE 
2.1 
Background Information 
Despite important advances over the past thirty years in the treatment of neonatal HSV disease, 
significant numbers of babies die or are left with lifelong neurologic sequelae.  Since the 1970s, 
the National Institute of Allergy and Infectious Diseases (NIAID) Collaborative Antiviral Study 
Group (CASG) has completed a series of antiviral treatment studies in neonates with HSV 
infection.  The first two of these trials involved vidarabine.2,3  These were followed by a 
comparative study between vidarabine and acyclovir in the 1980s,4 and then a trial of high dose 
acyclovir in the 1990s.5  Each of these investigations defined the standard of care for the 
management of babies with neonatal herpes, and decreased mortality from disseminated 
neonatal herpes from over 80% in the pre-antiviral era to 30% with high dose acyclovir 
therapy.2,5,6  Throughout the 2000s, the NIAID CASG conducted two placebo-controlled studies 
of oral acyclovir suppressive therapy following treatment of acute neonatal disease, which again 
redefined standard of care and simultaneously provided paradigm-shifting insights into the 
natural history of HSV infections of the central nervous system (CNS).7  In these suppression 
studies, babies with neonatal HSV CNS disease had significantly improved neurodevelopmental 
outcomes when immediately started on active antiviral suppression following acute disease 
compared with babies in whom oral antiviral suppression was deferred until after skin 
recurrences occurred or who only received placebo suppressive therapy.  These data indicate 
that subclinical viral replication with concomitant ongoing neurologic damage occurs during and 
following neonatal HSV CNS disease.  Even with immediate antiviral suppression, though, 
approximately three in 10 survivors of neonatal HSV CNS disease develop significant 
neurologic sequelae.7  Each year in the United States, over 100 babies die from neonatal 
herpes, and 250 survivors are left with significant neurologic damage.1,5,7-9  Approximately 1,000 
more survive without neurologic impairment, yet still require two to three weeks of 
hospitalization for intravenous therapy for acute disease, which puts significant emotional stress 
on their families – especially given the fact that this involves a sexually transmitted infection 
(STI) which the mother passed to her newborn.1,5,7  While the improvements in outcomes from 
the antiviral interventions defined by the CASG have been significant, the best mechanism of 
averting death, neurologic damage, and emotional distress from neonatal HSV is to prevent the 
disease from occurring in the first place. 
 
Clearly an ideal means of preventing neonatal HSV infection would be to prevent maternal 
genital infection with an effective vaccine.10  Since at least 20%, and more recently closer to 
50%, of genital herpes is caused by HSV-1,11 a successful vaccine would need to prevent both 
types of HSV.  No HSV-1 vaccine is in development, and the best HSV-2 vaccine candidate12 
recently failed to provide benefit in a large Phase III study.13  This trial, which represented a joint 

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effort between NIAID and GlaxoSmithKline (GSK), enrolled 8,323 women age 18-30 years at 50 
study sites in the United States and Canada and was conducted from 2002 to 2010.  Overall, 
the vaccine was not efficacious; vaccine efficacy was 20% (95% confidence interval [CI], −29 to 
50) against genital herpes disease.13  There are no other HSV-2 vaccine candidates that are in 
Phase II or III development, so vaccine prevention of maternal infection, and subsequent 
neonatal infection, is not likely for at least 15 years or more, if at all.  At a time when 
approximately 20% of all sexually active women in the United States have HSV-2 genital 
infection,14 and an additional 20%-50% of all genital herpes is caused by HSV-1,11 other means 
of preventing perinatal HSV transmission are urgently needed. 
 
Most babies developing neonatal herpes are delivered to women who are asymptomatically 
shedding HSV in their genital tract, have no identifiable signs or symptoms of active infection, 
and have no history of genital herpes.6,9,15  While maternal antiviral suppression is routinely 
provided near the end of pregnancy to women with a history of genital herpes,16-19 it is 
ineffective in completely suppressing viral shedding at delivery.18-21  Importantly, the CASG 
recently has reported cases of infants who developed neonatal herpes despite compliant 
maternal antiviral suppression.22,23  Furthermore, the approach of providing antiviral suppression 
to women with a history of genital herpes (classified as having “recurrent” infection) is 
intrinsically flawed, given that 60-80% of women delivering babies that develop neonatal HSV 
disease have no known history of genital herpes themselves.  Further, between 25% and 57% 
of women acquiring genital herpes during pregnancy (classified as having “first-episode primary” 
or “first-episode non-primary” infection) who are asymptomatically shedding at delivery will 
transmit the virus to their babies, compared with only 2% of women with recurrent genital 
herpes who are asymptomatically shedding at delivery.1,24-26  Thus, focusing on women with a 
history of genital herpes not only is ineffective, but also is illogical because these are not the 
women at highest risk of transmission of virus to their neonates. 
 
As noted above, since 85% of babies developing neonatal HSV disease acquire the virus from 
their mothers during the birth process,8 detecting which women are shedding HSV would allow a 
rational, targeted approach focusing on those neonates at risk.  Equally important, knowing that 
a woman with a history of genital herpes does not have HSV present in the genital tract at the 
time of delivery could have a positive impact on her obstetrical care by preventing unnecessary 
cesarean deliveries.  To accomplish this, however, information on the natural history of 
asymptomatic HSV shedding in the genital tract of pregnant women at the time of delivery is 
needed.  A simple, standardized, rapid test for detecting HSV in the maternal genital tract also is 
required.  PCR technologies have advanced to the point where this can now be considered.  A 
recent study demonstrated that real-time PCR detection of HSV when women are in labor is 
feasible.27  However, the assay used in that study required significant technical preparation of 
reagents and utilized multiple aliquoting steps on different machines to perform it correctly.  
Thus, this report served as proof of concept that real-time PCR detection in the labor and 
delivery suites is possible, but this assay is not a system which can be expanded to large-scale 
testing and utilization at multiple institutions. 

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An alternative approach is to use the GeneXpert PCR platform (Figure 
1) to rapidly and simply detect HSV in the genital tracts of women 
presenting to Labor and Delivery suites with the intent of delivery.  The 
GeneXpert system completely automates sample preparation, 
performing all the complex steps of DNA extraction in a single 
microfluidic cartridge (Figure 2).  Once the sample nucleic acid is 
extracted, it is moved in a completely automated fashion from the 
sample processing chamber in the cartridge into the reaction tube where amplification and 
detection of viral DNA take place (Figure 3).  All that is required to perform the test is to collect a 
genital swab and place it in viral transport media, and then to add approximately 1 mL of viral 
transport media to the Xpert HSV 1/2 Assay cartridge, close the 
cartridge cap, place the cartridge in the GeneXpert instrument, and 
press “start.”  The results then come off of the computer in about an 
hour. 
 
Significant parallels exist 
between the approach we 
will study in this staged trial 
and the prevention of 
neonatal disease caused by 
group B Streptococcus 
(GBS).  Approximately 25% 
to 30% of pregnant women 
have genital herpes,11,14 
compared with 10%-30% 
who are colonized with GBS in the vagina or rectum.28-
30  The incidence of neonatal herpes (0.33 cases per 
1,000 live births)1 is essentially identical to that of neonatal group B Streptococcal disease 
(0.34-0.37 cases per 1,000 live births).31  Both infections occur in neonates who are exposed to 
the pathogen in the mother’s vagina.  Colonization with GBS is intermittent,32,33 as is HSV 
shedding.34  An important difference, though, is that detection of GBS in women within 5 weeks 
of delivery serves as a marker of who will be colonized at delivery,29,35 whereas with HSV 
cultures obtained in the weeks prior to delivery do not predict which women will be shedding 
virus at delivery.36  Furthermore, recent data suggest that HSV may be intermittently shed in the 
genital tract for only a few hours at a time,37,38 further necessitating that testing for the virus 
occurs at the time of delivery. 
2.2 
Scientific Rationale 
With no HSV vaccine in the near- or intermediate-term future, reduction in cases of neonatal 
HSV disease requires improved identification of those babies at risk of neonatal infection.  To do 
Figure 1 
Fig 1.
 
Cartridge A – Valve Body
Port -
Direct Path
Output Port - Filter Path
Ultrasonic horn
Filter
Plunger in Syringe Barrel
Plunger stripping feature at 
top of syringe barrel
Glass Beads
Figure 2 
Figure 3 

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so on a large scale requires that the natural history of maternal genital shedding of HSV at 
delivery be fully understood and that methodologies exist to detect virus simply and rapidly.  The 
GeneXpert system provides a powerful opportunity to accomplish this with a test that is simple 
to perform and scalable to administer from a programmatic standpoint.  In this study, The Xpert 
HSV 1/2 Assay will be compared to routine PCR in both populations, and to viral culture in the 
symptomatic population.  The Xpert’s sensitivity and specificity will be determined in 
symptomatic nonpregnant women, and its positive and negative agreement will be determined 
in asymptomatic pregnant women.  All Xpert and routine PCR testing will be performed at the 
UAB Central Laboratory, with the Xpert PCR and routine PCR being performed in batches). 
The outcomes of neonates delivered to the 12,500 pregnant women enrolled on the current 
study (approximately half of whom will have virologic testing and neonatal outcomes recorded, 
and half of whom will have neonatal outcomes recorded and will have specimens banked for 
possible future virologic testing) will be queried and rates of neonatal HSV disease without 
preemptive therapy will be determined.  These incidence data will be compared with the 
incidence data from Brown et al.1  In their study of almost 60,000 women conducted over a 20 
year period, this group of researchers has reported an incidence rate for neonatal HSV disease 
of 1 in 3,200 live births. 
2.3 
Potential Risks and Benefits 
2.3.1 Potential Risks 
As a natural history and laboratory comparative study that does not include a therapeutic 
intervention, the potential risks relate to obtaining the vaginal sample or lesion swab, and having 
blood drawn.  For the vaginal (non-lesion) sampling, there is the possibility of minor discomfort 
of a swab in the vaginal vault.  For the lesion swab, there is a possibility also of minor discomfort 
in the area of the lesion.  For the blood sampling, there is the possibility of associated 
discomfort, bruising at the phlebotomy site, and rarely an infection. 
2.3.2 Known Potential Benefits 
For those women in Group 1, culture-confirmation of HSV infection will be provided to their 
clinicians.  For those women in Group 2, educational materials on neonatal HSV infection will be 
provided to the mothers. 

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3 
OBJECTIVES 
3.1 
Study Objectives 
3.1.1 
Primary Objectives 
 
• 
To estimate the sensitivity of the Xpert HSV 1/2 Assay relative to culture for 
detecting herpes simplex virus (HSV) DNA in the genital tract of nonpregnant 
women in sexually transmitted infections (STI) clinics 
• 
To estimate the positive percent agreement and negative percent agreement of 
the Xpert HSV 1/2 Assay relative to routine PCR for detecting HSV DNA in the 
genital tract of pregnant women admitted with the intent of delivery 
3.1.2 
Secondary Objectives 
 
• 
To estimate the positive predictive value between the Xpert HSV 1/2 Assay and 
culture for detecting HSV DNA in the genital tract of nonpregnant women in STI 
clinics  
• 
To estimate the positive percent agreement of the Xpert HSV 1/2 Assay relative 
to routine PCR for detecting HSV DNA in the genital tract of nonpregnant women 
in STI clinics 
• 
To compare the performance of Xpert with routine PCR in the combined group 
(pregnant and non-pregnant women) in detecting HSV-1 and HSV-2 virus, 
separately 
• To quantify HSV viral load, as determined by routine PCR, in the genital tract 
of women shedding the virus 
• To assess the type of infection (first-episode primary, first-episode non-primary, 
recurrent) among women shedding HSV 
 
3.1.3 
Exploratory Objectives 
• To determine rates of neonatal HSV disease among neonates delivered to 
pregnant women without active genital HSV lesions 
• To assess likelihood of viral transmission to neonates exposed to HSV during 
delivery as a manifestation of mode of delivery 
 

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3.2 
Study Outcome Measures 
3.2.1 Primary Outcome Measures 
• 
Sensitivity of detection of HSV DNA by Xpert HSV 1/2 Assay from specimens 
obtained from the genital tract of nonpregnant women in STI clinics as compared 
with HSV culture 
• 
Positive percent agreement and negative percent agreement of detection of HSV 
DNA by Xpert HSV 1/2 Assay from specimens obtained from the genital tract of 
pregnant women admitted with the intent of delivery, as compared with routine 
PCR detection of viral DNA 
3.2.2 Secondary Outcome Measures 
• Positive percent agreement of the Xpert HSV 1/2 Assay relative to routine PCR  
for detecting HSV DNA in genital tract of nonpregnant women in STI clinics 
• Positive predictive value of detection of HSV DNA by Xpert HSV 1/2 Assay from 
specimens obtained from the genital tract of nonpregnant women in STI clinics, 
as compared with culture 
• Comparison of Xpert and routine PCR results for detection of HSV-1 and HSV-2, 
separately 
• HSV viral load, as determined by routine PCR, in the genital tract of women 
shedding the virus 
• Correlation of HSV viral load, as determined by routine PCR, in genital tract of 
women with type of infection (first-episode primary, first-episode non-primary, 
recurrent) 
•  
3.2.3 
Exploratory Outcome Measures 
• HSV disease among neonates delivered to women without active genital HSV 
lesions 
• Correlation of HSV disease among neonates delivered to women without active 
genital HSV lesions and mode of delivery 
 
 
 

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4 
STUDY DESIGN 
The natural history of genital HSV shedding from the genital tract of pregnant and nonpregnant 
women will be evaluated, and the utility of a new cartridge-based PCR platform (the GeneXpert 
system with the Xpert HSV 1/2 Assay cartridge) for the detection of HSV-1 or HSV-2 will be 
assessed.  The study will be conducted at 15 academic medical centers throughout the United 
States.  Virologic assessments will be made in two populations:  1) nonpregnant women in STI 
clinics (likely at one or two study centers) with clinically-apparent genital HSV lesions (Group 1, 
n=300), and 2) pregnant women admitted with the intent of delivery (at all study centers) with no 
visible evidence of genital HSV infection (Group 2, n=12,500).  Women in each group will have 
specimens obtained from genital lesions (Group 1) or vaginal swabs (Group 2).  Specimens 
from all women in Group 1 will be evaluated by HSV culture, routine HSV PCR, and Xpert HSV 
1/2 PCR.  For both groups, Study Day 1 is the day that the genital or vaginal swab is obtained.  
Approximately half of the women (determined randomly) in Group 2 will be tested by routine 
HSV PCR and Xpert HSV 1/2 PCR; specimens from the rest of the women in Group 2 will be 
stored for possible testing in the future by routine HSV PCR and Xpert HSV 1/2 PCR.  In this 
manner, we will maximize the data from which to compare Xpert HSV 1/2 PCR results with 
routine PCR for women in Group 2, while maintaining flexibility to ensure an adequate number 
of specimens are positive for HSV DNA by routine PCR. 
All testing of samples on the GeneXpert platform and routine PCR in Group 1 and Group 2 will 
be performed in batches, while viral cultures performed in Group 1 will be performed in real time 
analysis.  All Xpert and routine PCR testing will be done at the UAB Central Laboratory.  In the 
conduct of this study, clinical staff may perform research activities (e.g., swabs for virologic 
testing, blood for serologic testing), under the direction of the site Principal Investigator or 
designee. 
Lesion swabs from women in Group 1 (non-pregnant women from STI clinics) will be placed in 
viral transport media, refrigerated at 4°C, and sent to the laboratory for processing.  The 
laboratory will perform real time analysis by HSV culture on a portion of the transport media, 
and freeze the remainder of the transport media in aliquots for subsequent batch testing by 
routine HSV PCR and Xpert HSV 1/2 PCR at the UAB Central Laboratory. 
Vaginal swabs from women in Group 2 (pregnant women admitted with the intent of delivery) 
will be placed in viral transport media, frozen at -20°C or colder, and batch-shipped to the UAB 
Central Laboratory for future PCR analysis by routine HSV PCR and Xpert HSV 1/2 PCR.  
Specimens from approximately half of the Group 2 women will be analyzed by routine PCR.  All 
specimens evaluated by routine PCR will also be evaluated by Xpert HSV 1/2 PCR.  The 
laboratory personnel performing each type of procedure (routine PCR for Groups 1 and 2; 
culture for Group 1; and Xpert HSV 1/2 PCR for Groups 1 and 2) will be masked to the results of 
the other tests for a given subject.  A blood specimen will be obtained from each nonpregnant 

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(Group 1) and pregnant (Group 2) woman on Study Day 1, and if she is determined to be 
shedding HSV by routine PCR, Xpert HSV 1/2 PCR, or culture then type-specific serologic 
testing will be performed.  All virologic and serologic testing in this protocol is for research and 
not for standard of care.  Correlation of viral typing from the virologic sampling with HSV-1 and 
HSV-2 serostatus will allow for categorization of infection (first-episode primary, first-episode 
nonprimary, or recurrent infection). 
HSV culture results from women in Group 1 will be reported to their treating physician.  Virologic 
results from women in Group 2 will not be reported back to the subject or her physician.  The 
reasons for not reporting results to the Group 2 subject or her physician are two-fold:  1) the 
PCR analyses are performed in batches, so no results will be available within a timeframe in 
which clinical decisions on the woman’s care could be made: and 2) neither routine HSV PCR 
nor Xpert HSV 1/2 Assay are FDA-cleared for evaluation of HSV testing on women without 
active lesions, and thus the reliability of a positive or a negative test result in this population is 
not known.  Even if PCR results (routine HSV PCR or Xpert HSV 1/2 Assay) could be obtained 
in a real-time (non-batched) fashion, it would be irresponsible to report results from a non-
validated test to the subject and her health care provider, as they may be influenced to make 
clinical decisions based on this unvalidated information. Furthermore, using the results of this 
research test for clinical decision making is not the intent of this study protocol.  All pregnant 
women in Group 2 will receive written materials on Study Day 1 educating them on signs and 
symptoms of neonatal HSV disease.  All postpartum women or designated representative will 
be contacted 30-90 days post-delivery and an inquiry will be made to determine mode of 
delivery and whether their babies developed neonatal HSV disease. 
Data on the incidence of neonatal HSV disease among babies delivered to women in Group 2 
will be compared with the incidence data from Brown et al.1  In their study of almost 60,000 
women conducted over a 20 year period, this group of researchers has reported an incidence 
rate for neonatal HSV disease of 1 in 3,200 live births. 
Study involvement for women in Group 1 will be limited to only 1 day on Study Day 1 (day of 
virologic sampling).  Women in Group 2 will be involved in the study for up to 90 days, with 
virologic sampling obtained and educational materials provided on Study Day 1, and with follow-
up contact performed between Study Day 30 and Study Day 90.  Full study accrual will occur 
within 4 years of enrollment of the first study subject. 
4.1 
Substudies (if applicable) 
Not Applicable 

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5 
STUDY POPULATION 
Enrollment will be confined to women 18 years of age and older who are nonpregnant and have 
active genital HSV lesions (Group 1, n=300) or women 19 years of age and older who are 
pregnant and do not have visible genital HSV lesions or prodromal symptoms (Group 2, 
n=12,500).  Women of all races will be included. 
5.1 
Recruitment Strategies 
Identification of study subjects will be done based on the local standards of practice and IRB 
approved means.  Clinicians who are care providers for these patients will identify patients who 
would generally qualify for inclusion in Group 1 or Group 2 of the study and will confirm that it is 
acceptable by the patient for the research team to approach them to discuss inclusion in the 
study.  Group 1 subjects will be recruited from the STI clinic at the Health Department and/or 
similar outpatient facility.  Group 2 subjects will be recruited from labor and delivery facilities 
and/or identified through obstetric practices.  Subjects can sign informed consent in the labor 
and delivery facilities, or can sign informed consent prior to that time if the woman is 34 weeks 
estimated gestational age or older.  However, no study related samples/procedures can be 
initiated until subject is admitted to the hospital labor and delivery area with the intent of 
delivering her baby.  Each participating facility will determine the appropriate setting from which 
to recruit subjects.  IRB approval will be obtained for each site’s recruitment plan.  Site 
investigators (who may also be the treating clinician) or study coordinators will provide 
information about the intended study population to the clinicians to aid in identifying appropriate 
patients to be approached for inclusion and consent.  Consenting will be conducted by the 
research team (e.g., investigator and/or study coordinator/trained designee) for Group 1 and 
Group 2.  Informed consent will be obtained prior to any study required procedures.  The State 
of Alabama has recently (2015) changed the age of consent from 19 years to 18 years.  As a 
result, Group 1 (which is still enrolling at the time of amendment of the protocol to version 4.0) 
will now enroll women who are 18 years of age or older.  Recruitment and enrollment into Group 
2 is not affected by this change because the cohort is already fully enrolled. 
5.2 
Subject Inclusion Criteria 
• 
Signed Informed Consent from woman 
• 
≥ 18 years of age (Group 1); ≥ 19 years of age (Group 2) 
• 
Female gender 

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• 
Group 1: women not known to be pregnant and being evaluated in STI clinics for herpetic 
genital lesions; OR Group 2:  pregnant women ≥ 34 weeks estimated gestational age 
5.3 
Subject Exclusion Criteria 
• 
For women in Group 2 (pregnant women admitted with the intent of delivery), active herpetic 
lesions or prodromal symptoms in the genital region 
• 
Receipt of acyclovir, valacyclovir, or famciclovir within the previous 14 days 
• 
Known HIV infection 

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6 
STUDY PROCEDURES/EVALUATIONS 
6.1 
Test Device 
The Xpert HSV1/2 Assay, performed on the GeneXpert Instrument Systems, is a new qualitative 
in vitro PCR test for the automated and rapid detection and qualitative differentiation of HSV 1 
and HSV 2 in clinician-collected genital swabs.  The Xpert HSV1/2 Assay system performs 
multiplex polymerase chain reaction (PCR) for detection of DNA targets after an initial sample 
processing step.  The system consists of a GeneXpert® instrument, personal computer, and 
disposable fluidic cartridges that are designed to complete sample preparation and real-time 
PCR for detection of HSV1 and/or HSV2 in about 1 hour. 
The single-use cartridges contain:  1) eleven chambers for holding sample, reagents, or other 
materials; 2) a valve body composed of a plunger and syringe barrel; 3) a rotary valve system 
for controlling the movement of fluids between chambers; 4) an area for capturing, 
concentrating, washing, and lysing of virus; 5) dry, real-time PCR reagents; 6) liquid reagents 
for PCR; and 7) an integrated PCR reaction tube that can be automatically filled by the 
instrument.  To eliminate test-to-test contamination, all fluids including amplicons are contained 
within the disposable cartridge.  The instrument never comes into contact with any fluids within 
the cartridge.  Each cartridge is intended to test one sample.   
The primers and probes in the Xpert HSV1/2 Assay detect and differentiate HSV-1 and HSV-2 
DNA sequences.  Each test cartridge includes a Sample Processing Control (SPC), a Sample 
Adequacy Control (SAC), and a Probe Check Control (PCC).  The SPC is present to control for 
adequate processing of the target bacteria and to monitor the presence of inhibitors in the PCR 
reaction.  The SAC reagents detect the presence of a single copy human gene and monitor 
whether the sample contains human DNA.  The PCC verifies reagent rehydration, PCR tube 
filling in the cartridge, probe integrity, and dye stability. 
Cepheid (Sunnyvale, CA) will provide the GeneXpert system (instrument and cartridges).  Xpert 
HSV 1/2 Assay cartridges will be appropriately labeled in accordance with regulations, and will 
be stored as indicated in the package insert.  The UAB Central Laboratory personnel will be 
responsible for maintaining logs of receipt, accountability, storage conditions of the Xpert HSV 
1/2 Assay investigational device and associated records and results.  Documents and the 
investigational device will be stored in a secure and accessible location. 
6.2 
Study Procedures 
If a patient is eligible for the study based on information regarding inclusion/exclusion criteria 
that is collected as part of the standard medical care (and not obtained for study purposes), then 

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the clinical team, following consultation with the patient indicating that she is interested to hear 
more about the study, will contact the study staff who will approach the patient and initiate the 
informed consent process (Section 11).  For Group 1, signing of the informed consent document 
will take place on the same day as study procedures.  For Group 2, signing of the informed 
consent document may occur on or before Study Day 1.  Patients may be provided with study 
information prior to confirmation of eligibility for enrollment.  After consent is obtained, the study 
participant will begin participation in the study procedures. 
Following informed consent and confirmation of eligibility criteria, virologic sampling will be 
obtained from women in Group 1 and Group 2.  No laboratory tests will be done to confirm 
eligibility.  For the Group 1 women (nonpregnant women with active genital HSV lesions), a 
swab of the lesion(s) will be obtained and placed in viral transport media.  For the Group 2 
women (pregnant women without active lesions), following admission to the hospital labor and 
delivery area with the intent of delivering her baby a swab of the vaginal vault/canal will be 
obtained and placed in viral transport media; utilization of a speculum examination is not 
required in this sampling process.  The specimen should be obtained before delivery, but may 
be obtained following delivery as long as it is collected within 24 hours following delivery.  
Women in both Groups 1 and 2 will have a venipuncture for collection of blood for serologic 
testing.  This test will be performed only if the subject’s virologic studies identify HSV shedding. 
Women in Group 2 will be provided with printed material describing neonatal HSV signs and 
symptoms.  These women (Group 2) or their designated representative also will be contacted 
between Study Day 30 and Study Day 90 for self-reporting of the delivery mode (vaginal vs. 
Cesarean delivery) and the development of neonatal HSV symptoms and signs post-delivery.  It 
is acceptable to initiate contact shortly following the 30 day window and multiple attempts may 
be needed to reach the mother or her designated representative.  The initial maternal Informed 
Consent document will include obtaining parental permission for the neonate in order to ask 
these questions.  Verbal reporting from the mother on maternal receipt of antiviral therapy at 
any point following enrollment also will be captured from all women in Group 2. 
HSV culture results (not PCR) from women in Group 1 will be reported to their treating 
physician.  Virologic results from women in Group 2 will not be reported back to the subject or 
her physician.  The reasons for not reporting results to the Group 2 subject or her physician are 
two-fold:  1) the PCR analyses are performed in batches, so no results will be available within a 
timeframe in which clinical decisions on the woman’s care could be made: and 2) neither routine 
HSV PCR nor Xpert HSV 1/2 Assay are FDA-cleared for evaluation of HSV testing on women 
without active lesions, and thus the reliability of a positive or a negative test result in this 
population is not known.  Even if PCR results (routine HSV PCR or Xpert HSV 1/2 Assay) could 
be obtained in a real-time (non-batched) fashion, it would be irresponsible to report results from 
a non-validated test to the subject and her health care provider, as they may be influenced to 
make clinical decisions based on this unvalidated information. Furthermore, using the results of 
this research test for clinical decision making is not the intent of this study protocol. 

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Subjects are not required to be seen in person after their involvement on Study Day 1.  
However, for Group 2 study staff may visit or contact the subject before she is discharged from 
the hospital to confirm the follow-up contact information.  No additional contact is made with the 
subject following Study Day 1 for Group 1, and following the Study Day 30-90 contact for Group 
2. 
6.3 
Laboratory Evaluations 
6.3.1 Laboratory Evaluations/Assays 
Virologic Testing 
Samples obtained from women in Group 1 and Group 2 will be tested in batches for HSV DNA 
by Xpert HSV 1/2 PCR and routine PCR.  Additionally, samples from women in Group 1 will be 
tested in real time by HSV culture.  In this trial, all of these virologic tests will be Research 
Laboratory Evaluations (i.e., they would not be performed if the subject were not enrolled in the 
study).  Routine HSV PCR from genital mucosa is not an FDA-cleared test.  All Xpert and 
routine PCR testing will be conducted at the UAB CLIA-certified Central Laboratory.  Bi-
directional sequencing will be performed on specimens with discordant results between the 
Xpert HSV 1/2 PCR and routine PCR from asymptomatic pregnant women admitted with the 
intent of delivery, and between Xpert HSV 1/2 PCR and HSV culture in nonpregnant women.  
The primers used for bi-directional sequencing will be different from those used in the Xpert 
HSV 1/2 PCR and the routine PCR methods.  Note: The results of discrepant analysis are for 
informational purposes only and will not be used to recalculate the performance of the Xpert 
HSV 1/2 PCR Assay. 
Venipuncture 
Women in both Groups 1 and 2 will have a venipuncture on Study Day 1 for collection of at least 
0.5 mL of serum for serologic testing for HSV type-specific antibody.  This serologic test will be 
performed for subjects in either group only if the subject’s virologic studies identify HSV 
shedding.  In this trial, all serologic tests will be Research Laboratory Evaluations (i.e., they 
would not be performed if the subject were not enrolled in the study).  The window of time for 
collection of this specimen from women in Group 2 is ± 3 days.  Remnant blood samples from 
clinical laboratory draws may be retrieved for this serologic testing.  Informed consent will be 
obtained prior to any study required procedures. 

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6.3.2 Specimen Preparation, Handling, and Shipping 
6.3.2.1 
Instructions for Specimen Preparation, Handling, and Storage 
Group 1:  Lesion swabs from nonpregnant women in STI clinics will be placed in viral transport 
media, refrigerated at 2-8°C, and transported by the study coordinator or designee in real time 
to the laboratory for analysis by HSV culture in real time, and by Xpert HSV 1/2 PCR and 
routine HSV PCR (batched analysis at the UAB Central Laboratory). 
Group 2:  Vaginal swabs from pregnant women admitted with the intent of delivery will be 
placed in viral transport media, frozen at -20°C or colder, and batch-shipped to the UAB Central 
Laboratory for batched analysis by Xpert HSV 1/2 PCR and by routine HSV PCR.  Only one 
freeze-thaw cycle will be required for each type of PCR test.  Specimens will be assigned by the 
Study Biostatistician to either batched processing by routine PCR or to storage for possible 
testing by PCR in the future.  Those that are batch processed by routine PCR also will be 
analyzed subsequently by Xpert HSV 1/2 PCR. 
Serum from blood samples will be frozen at -20°C or colder and batch-shipped to the UAB 
Central Laboratory for HSV serologic testing.  If remnant blood is retrieved for this purpose, it 
should have been stored according to local laboratory standard operating procedures until 
retrieval for research purposes. 
Specific instructions on specimen preparation, handling, and shipping will be provided in the 
Manual of Procedures (MOP) for this study.  Residual specimens will be stored at the UAB 
Central Laboratory if consent is provided by the study subject.  Access to these specimens is 
detailed in Section 10.1. 
6.3.2.2 
Specimen Shipment 
Specific instructions on specimen shipments, including frequency and location, will be provided 
in the Manual of Procedures (MOP) for this study. 

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7 
STATISTICAL CONSIDERATIONS 
7.1 
Study Hypotheses 
For pregnant women, we hypothesize that the long term expected positive percent agreement of 
HSV DNA using the Xpert HSV 1/2 Assay is 95% as evidenced by confidence interval 
containing this value.  Therefore, with a sample size of between 135 and 150 we will have over 
a 95% probability of obtaining a point estimate greater than 91.8% with a lower bound of a 95% 
CI greater than 85%.  This can be summarized in the form of hypothesis testing as: 
 
H0: Positive Percent Agreement = 95% 
HA: Positive Percent Agreement < 85% 
Sample Size 135-150 
Critical Value = 11FN, (or point estimate > 91.85% with lower 95% CI > 85%) 
Target Alpha Value = 5%, Power > 95%.  
 
For non-pregnant women, we hypothesize that the long term expected sensitivity of HSV DNA 
using the Xpert HSV 1/2 Assay is 95.34% as evidenced by a 95% confidence interval containing 
this value.  Therefore, with a sample size of between 270 and 300 we will have a 95% 
probability of obtaining a point estimate greater 93.3% with a lower bound of a 95% CI greater 
than 90%.  This can be summarized in the form of hypothesis testing as: 
 
H0: Sensitivity = 95.34% 
HA: Sensitivity < 90% 
Sample Size 270-300 
Critical Value = 18 false negative (FN), (or point estimate > 93.3% with lower 95% CI > 89%) 
Target Alpha Value = 5%, Power > 95%. 
7.2 
Sample Size Considerations 
Approximately 6,000 pregnant women (of the 12,500 enrolled) admitted with the intent of 
delivery will be tested to detect 144 (2.4%) who are shedding HSV by PCR.  A subject who 
enrolls on the study but does not have a vaginal specimen obtained will not count toward the 
target sample size.  The process of determining the 6,000 to be tested by PCR is as follows:  
IDs of pregnant women enrolled in a given 3 month period (each quarter) will be randomly 
assigned by site to either (1) the group to be tested by PCR or (2) the group not to be tested by 
PCR.  This will enable us to have better representation over time and across sites.  Specimens 
will be collected and tested from an additional 300 nonpregnant women presenting to STI clinics 
with active herpetic lesions who are shedding HSV by both PCR and culture.  A 95% confidence 
interval will be constructed for the sensitivity of Xpert HSV 1/2 Assay relative to culture for the 
nonpregnant women population.  Since we do not anticipate many negative results in the 

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symptomatic nonpregnant population, we may not be able to estimate the specificity of the 
GeneXpert in this population.  For the pregnant women population, 95% confidence intervals 
will be constructed for the true positive and negative percent agreement between Xpert and 
routine PCR.  The sample size justification considers the width and lower bound of these 95% 
confidence intervals for a single proportion using Clopper-Pearson method (also known as the 
Fisher’s Exact CI) for small samples and large-sample confidence intervals for samples of 
greater than 100 (Ref Nonparametric Statistical Methods, Hollander and Wolf, Wiley, NY, NY 
1973).  SAS version 9.2 was used for these calculations. 
Table 1 displays the minimum number of Xpert HSV 1/2 Assay positives from the total number 
of routine PCR positives in order to get a lower bound of a 95% confidence interval of at least 
86%. With the expected 144 routine PCR positives from the pregnant women admitted with the 
intent of delivery, if the Xpert HSV 1/2 Assay finds 133 positives, then the corresponding 95% 
confidence interval for the true positive percent agreement will have a width of 9.4% and the 
resulting interval estimate is [86.7%, 96.1%]. Similar interpretations may be applied to the other 
cases considered in this table.  With an expected 5856 negatives out of 6000 pregnant women, 
the 95% confidence interval for the negative percent agreement will be very tight. In particular, if 
the observed negative percent agreement is 97% (i.e., Xpert HSV 1/2 Assay finds 5680 
negatives out of the 5856 routine PCR negatives), then the corresponding 95% confidence 
interval for the true negative percent agreement of Xpert HSV 1/2 Assay and routine PCR will 
have a width of 0.9% and the resulting interval estimate is [96.5%, 97.4%]. The reason for using 
positive and negative percent agreement instead of sensitivity and specificity is that routine PCR 
is not considered a gold standard. 
Table 1: Minimum number of  Xpert HSV 1/2 Assay positives, corresponding 95% Confidence 
Interval (CI) for Positive Percent Agreement, and width of the interval 
Number of PCR 
Positives 
135 
137 
139 
142 
144 
150 
Number of Xpert 
HSV 1/2 Assay   
Positives 
124 
126 
128 
131 
133 
138 
Greatest number 
of Xpert HSV 1/2 
Assay   FN 
Allowed 
11 
11 
11 
11 
11 
12 
95% CI for 
positive % 
agreement 
85.9, 95.9 
86.1, 95.9 
86.3,96.0 
86.6, 96.1 
86.7, 96.1 
86.4, 95.8 
Width of CI 
10.0 
9.8 
9.9 
9.5 
9.4 
9.4 
For nonpregnant women presenting to STI clinics with active herpetic lesions, Table 2 displays 
the minimum number of Xpert HSV 1/2 Assay positives from the total number of culture 
positives out of 300 subjects in order to get a lower bound of a 95% confidence interval of 
approximately 90%.  Since we conservatively expect 270 of the 300 (90%) to be shedding HSV 
by culture, this is the smallest number of positive cultures considered in Table 2.  To interpret, if 

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Xpert HSV 1/2 Assay finds 252 positives out of the 270 culture positives, the corresponding 
95% confidence interval for the true sensitivity will have a width of 6.3% and the resulting 
interval estimate is [89.7, 96.0%].  Similar interpretations may be applied to the other cases 
considered in this table.  The specificity of Xpert HSV 1/2 Assay may not be estimated in this 
population because we do not expect many culture-negative women.  The main purpose of 
including this population is to enable us to get a precise estimate of the sensitivity of Xpert HSV 
1/2 Assay relative to viral culture in women with symptomatic HSV lesions. 
Table 2: Minimum number of positives and corresponding 95% Confidence Interval (CI) for 
Sensitivity 
Number of 
Culture 
Positives 
270 
275 
280 
285 
290 
295 
300 
Number of 
Xpert HSV 1/2 
Assay   
Positives 
252 
257 
261 
266 
271 
276 
280 
Greatest 
number of Xpert 
HSV 1/2 Assay 
FN Allowed 
18 
18 
19 
19 
19 
19 
20 
95% CI for 
sensitivity 
89.7, 96.0 
89.9, 96.1 
89.6, 95.9 
89.8, 95.9 
90.0,96.0 
90.1, 96.1 
89.9, 95.9 
Width of CI 
6.3 
6.5 
6.3 
6.1 
6.0 
6.0 
6.0 
 
7.3 
Participant Enrollment and Follow-Up 
Subjects will be enrolled in one of two groups based upon their clinical status.  Group 1 (n=300) 
will consist of women with clinically apparent herpetic genital lesions in STI Clinics.  Group 2 
(n=12,500) will consist of women without clinically apparent genital HSV lesions who are 
admitted with the intent of delivery.  For women in Group 2, all study procedures will be uniform 
at each study site; once specimens from a given subject are received in the UAB Central 
Laboratory, the Study Biostatistician will randomly assign them to either batched processing by 
routine PCR or to storage for possible testing by PCR in the future.  Those that are batch 
processed by routine PCR also will be analyzed subsequently by Xpert HSV 1/2 PCR.  We 
anticipate that approximately half of women enrolled in Group 2 will have batched PCR analysis 
of their specimens performed, with the other half having their specimens stored. 
Subjects in Group 1 and Group 2 are not seen in person after their involvement on Study Day 1.  
HSV culture results from women in Group 1 will be reported to their treating physician.  Women 
in Group 2 or their designated representative will be contacted between Study Day 30 and 
Study Day 90 to assess for neonatal treatment for HSV infection and for maternal mode of 
delivery. 

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7.4 
Analysis Plan 
Analyses of Group 1 and Group 2 may occur at different times, if Group 1 (n=300) fully accrues 
earlier than Group 2 (n=12,500).  Demographics and clinical characteristics will be summarized 
using means and standard deviations for continuous variables and counts and proportions for 
categorical variables. For continuous variables and chi-squared tests for categorical variables, 
ANOVA (or its nonparametric analog Kruskall-Wallis) will be utilized to compare these 
characteristics among groups of participants.  
For pregnant women, the primary goal of this study is to estimate the positive and negative 
percent agreement of the Xpert HSV 1/2 Assay relative to routine PCR methods. For 
nonpregnant women, we will be able to estimate the sensitivity of the Xpert HSV 1/2 Assay 
relative to culture.  As a secondary analysis, we will also estimate the positive percent 
agreement of the Xpert HSV 1/2 Assay relative to PCR for this population.  For all these 
quantities, we will utilize the exact Clopper-Pearson method for small sample and large-sample 
methods for samples greater than 100 to construct 95% confidence intervals for a single 
proportion.  To compare the performance of Xpert with routine PCR for the combined groups 
(pregnant and non-pregnant women) in detecting HSV-1 and HSV-2 virus, we present the 
results of all samples tested in a 2x2 table separately for each type of virus. We will perform 
McNemar’s test and construct 95% confidence interval estimates for the positive and negative 
percent agreement for each type of virus. 
Since it is also of interest to estimate the true proportion of babies developing HSV infection 
from the pregnant women participants, we also will construct a confidence interval for the true 
proportion using either exact or large-sample methods. 

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8 
SOURCE DOCUMENTS AND ACCESS TO SOURCE 
DATA/DOCUMENTS 
Documentation of source data is necessary for the reconstruction, evaluation, and validation of 
clinical findings, observations, and other activities during a clinical trial.  Source documentation 
serves to substantiate the integrity of the trial data, to confirm observations that are recorded, 
and to confirm the existence of study participants.  This standard also serves to ensure data 
quality by creating audit trails and enabling verification that data are present, complete, and 
accurate.  This study will be monitored using these standards. 
The site will maintain appropriate medical and research records for this trial, in compliance with 
ICH E6, Section 4.9 and regulatory and institutional requirements for the protection of 
confidentiality of subjects.  As part of participating in a DMID-sponsored, DMID-affiliated, or 
manufacturer-sponsored study, the site will permit authorized representatives of the sponsor(s), 
DMID, the UAB Central Unit, and regulatory agencies to examine (and when required by 
applicable law, to copy) clinical records for the purposes of quality assurance reviews, audits, 
and evaluation of the study safety and progress. 
Examples of these original documents and data records may include, but are not limited to, 
Source Document Worksheets, hospital records, clinical and office charts, recorded data from 
automated instruments, and subject files and records kept at the laboratories. 
Sites that are participating in this trial should consult the MOP and DMID/NIAID Source 
Document Standards (most current version) for specific instruction and forms. 
The study monitor or other authorized representatives of the sponsor, or other governmental 
regulatory agencies may inspect all documents and records required to be maintained by the 
investigator for this trial.  The clinical study site will permit access to such records. 

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9 
QUALITY CONTROL AND QUALITY ASSURANCE 
The study site will implement a quality management plan.  The quality management procedures 
are described herein, as well as in the Manual of Procedures and the study site quality 
management plan (QMP).  Per the QMP, data will be evaluated for compliance with the protocol 
and accuracy in relation to source documents.  The study will be conducted in accordance with 
procedures identified in the protocol.  Items to be reviewed include, but are not limited to: 
eligibility (including informed consent), any required AE reporting, study/clinical endpoints, 
follow-up visits, regulatory documents, missed calls, and review of clinical records.  Data that 
will be reviewed, who is responsible for implementation, and the schedule for internal reviews 
will be specified or referenced in the quality management plan. 
The UAB Statistical and Data Coordinating Center will implement quality control procedures 
beginning with the data entry system, and will generate data quality control checks that will be 
run on the database within 24 hours of new data entered into the system.  Full documentation of 
these checks will be provided to the UAB Central Unit so that any resulting queries can easily 
be understood and transmitted to the respective site for resolution within a short period of time.  
These processes are validated by a second programmer and also tested with faulty test data 
(refer to Section 13 for additional details). 
Site monitoring will also be implemented by a DMID directed independent monitoring group.  
See Section 12 (Clinical Monitoring Structure) for additional details. 

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10 
SUBJECT CONFIDENTIALITY 
The information obtained during the conduct of this clinical study is confidential, and disclosure 
to third parties other than those noted below is prohibited.  The results of the research study 
may be published, but study participant’s names or identities will not be revealed.  Records will 
remain confidential.  To maintain confidentiality, the principal investigators at each site will keep 
records in locked cabinets and the results of tests will be coded to prevent association with 
volunteers’ names.  Data entered into computerized files will be accessible only by authorized 
personnel directly involved with the study and will be encoded.  Data received by DMID and 
forwarded to Cepheid will not include subject specific data but only encoded data.  However, 
subject specific information will be available to the clinical monitors, and to health authorities 
where provided by law.  
 
The study investigator is obliged to provide the NIAID/DMID and the UAB Central Unit with 
complete test results and all data developed in this study.  The NIAID/DMID or the UAB Central 
Unit may disclose this information to appropriate regulatory authorities or clinical practice 
management groups (such as Pediatric Infectious Disease Society) as deemed necessary by 
the NIAID/DMID or the UAB Central Unit.   
  
Subject-specific information may be provided to other appropriate medical personnel only with 
the study participant’s permission.  To ensure compliance with current ICH guidelines, data 
generated by this study must be available for inspection upon request by representatives of 
national and local health authorities, NIAID/DMID, the UAB Central Unit, and the Central 
Facilitated IRB/IEC overseeing the study (or for each study site, if the site does not utilize the 
Central Facilitated IRB). 
10.1 Future Use of Stored Specimens 
Approximately half of the 12,500 women enrolled in Group 2 will have their specimens stored for 
possible future HSV testing by PCR and serology.  This will be fully explained in the Informed 
Consent document and will not be subject to “Future Use of Specimens” documentation since 
this testing is the primary purpose of the study.  Likewise, any additional comparator PCR 
assays for HSV DNA that might be required by the FDA for this study will not be subject to 
“Future Use of Specimens” documentation since this comparison is the primary purpose of the 
study. 
Subjects will be asked at the time of enrollment for permission to keep any remaining 
specimens for possible future use.  Some of the specimens obtained from study participants 
during this study will be stored indefinitely in the UAB Central Laboratory at the University of 
Alabama at Birmingham and may be used in future viral or other sexually transmitted infection 

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research.  These specimens will be labeled with a code number and not with the study 
participant’s name.  At the time of consent for study participation, study participants will have the 
opportunity to either agree to have their specimens used in future research or decline to have 
their specimens used in future research.  The study participant will indicate her preference by 
initialing the appropriate line or checking the appropriate box of the Consent Form in the section 
entitled, “Future Use of Specimens”.  Non-protocol designated, future testing of samples will be 
performed only on samples from study participants who have consented for future testing of 
samples. 
Samples may be shared with other investigators at other institutions or with the manufacturer of 
the GeneXpert cartridge.  The samples will not be sold or used directly for production of any 
commercial product.  No human genetic tests will be performed on samples.  Long term storage 
of residual samples will be established according to OHRP guidelines ensuring that codes or 
other personally identifying links will not be distributed to future researchers. 
The specimens will be stored indefinitely in the UAB Central Virology Laboratory at the 
University of Alabama at Birmingham.  Specimens from study participants will be labeled and 
coded without study participant’s identifiers.  If the study participant has indicated in the signed 
consent form that she does not agree to allow the future use of specimens for future research, 
then her specimens will be destroyed at the completion of the study. 

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11 
INFORMED CONSENT PROCESS 
The process of obtaining informed consent must be documented in the medical records, clinic 
chart, and/or research chart.  The consent form must be signed and dated by the study 
participant before participation in the study.  A copy of the signed consent form must be 
provided to the study participant.  Signed consent forms must remain in each study participant’s 
study file and must be available for verification by study monitors at any time.  
 
The investigational nature and research objectives of this trial, the procedure, and its attendant 
risks and discomforts will be carefully explained to the study participant.  A signed informed 
consent document will be obtained from each study participant prior to entry into this study.  
This informed consent document will describe the study-related procedures for the woman, and 
for women in Group 2 it also will include permission to contact them at 30 to 90 days to inquire 
about the status of their baby and, if necessary, to inspect the infant’s medical chart.  Waiver of 
Assent (or obtaining parental permission) will be provided at the time the signature is obtained 
on the Informed Consent document (see Section 11.1).  At any time during participation in the 
protocol, if new information becomes available relating to risks, this information will be provided 
orally or in writing to all enrolled or prospective study participants. Documentation will be 
provided to the Central Facilitated IRB/IEC overseeing the study (or to each study site, if the site 
does not utilize the Central Facilitated IRB) and, if necessary, the informed consent will be 
amended to reflect any relevant information. 
 
There will be two consent forms, one for Group 1 and one for Group 2.  The informed consent 
will reflect that the HSV culture results from women in Group 1 will be reported to their treating 
physician, and that virologic results from women in Group 2 will not be reported back to the 
subject or her physician.  The reasons for not reporting results to the Group 2 subject or her 
physician are two-fold:  1) the PCR analyses are performed in batches, so no results will be 
available within a timeframe in which clinical decisions on the woman’s care could be made: and 
2) routine HSV PCR and Xpert HSV 1/2 Assay are not FDA-cleared tests, so at this point the 
reliability of a positive or a negative test result is not known.  Even if PCR results (routine HSV 
PCR or Xpert HSV 1/2 Assay) could be obtained in a real-time (non-batched) fashion, it would 
be irresponsible to report results from a non-validated test to the subject and her health care 
provider, as they may be influenced to make clinical decisions based on this unvalidated 
information. Furthermore, using the results of this research test for clinical decision making is 
not the intent of this study protocol. 
An investigator shall seek such consent only under circumstances that provide the prospective 
subject sufficient opportunity to consider whether or not to participate and that minimize the 
possibility of coercion or undue influence. The information that is given to the subject shall be in 
language understandable to the subject or the representative. 

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Subjects will sign the informed consent document prior to any procedures being done 
specifically for the study.  Subjects should have the opportunity to discuss the study with their 
family, friends or personal physician, or think about it prior to agreeing to participate.  Subjects 
may withdraw consent at any time throughout the course of the trial.  A copy of the informed 
consent document will be given to the subjects for their records.  The rights and welfare of the 
subjects will be protected by emphasizing to them that the quality of their medical care will not 
be adversely affected if they decline to participate in this study. 
11.1 Informed Consent/Assent Process (in Case of a Minor or Others 
Unable to Consent for Themselves) 
The site principal investigator will ensure that this study is conducted in full conformity with 
principles of the Belmont Report:  Ethical Principles and Guidelines for the Protection of Human 
Subjects of Research of the National Commission for the Protection of Human Subjects of 
Biomedical and Behavioral Research (April 18, 1979) and codified in 45 CFR 46, 21 CFR 50 
and 56, and ICH E6; 62 Federal Regulations 25691 (1997), if applicable.  The site principal 
investigator’s Institution will hold a current Federal Wide Assurance (FWA) issued by the Office 
of Human Research Protection (OHRP) for federally funded research. 
For women in Group 2, parental/legal guardian permission will be obtained for the minor’s (the 
subject’s baby) information to be gathered on or between Study Days 30-90.  Assent age will be 
determined by the Central Facilitated IRB/IEC overseeing the study (or by each study site, if the 
site does not utilize the Central Facilitated IRB).  Appropriate documentation will be required for 
subjects who are the age of assent, whether mature enough to read and capable of providing 
signed assent, or whether too young to read but capable of providing verbal assent, as 
determined by the IRB in compliance with 45CFR46.408.  The Central Facilitated IRB/IEC 
overseeing the study (or each study site, if the site does not utilize the Central Facilitated IRB) 
will review and assign the risk level. 
11.2 Institutional Review Board 
Reviewing IRBs will be registered with the OHRP and have Federal Wide Assurance (FWA) to 
conduct US federally-funded studies. 
Prior to enrollment of subjects into this trial, the sponsor’s approved protocol, informed consent 
documents, relevant supporting information, and all types of volunteer recruitment, 
advertisements, and information provided to subjects (summary, pamphlets, etc.) will be 
reviewed and approved by the appropriate IRB listed on the institution’s FWA.  
In addition, the IRB/REC will review and approve this protocol in compliance with applicable US 
federal regulations for the protection of pregnant women, human fetuses, and neonates in this 
study. 

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The responsible official for the IRB will sign the IRB letter of approval of the protocol prior to the 
start of this trial and a copy will be provided to the UAB Central Unit, who will then provide a 
copy to DMID.   
Should amendments to the protocol be required, the amendments will be approved by the 
sponsor and provided by the UAB Central Unit to the site principal investigator for submission to 
the IRB. Any amendments to the protocol or consent materials must be approved by the 
IRB/REC before they are placed into use, unless for immediate safety to the subject. 
The 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 per 
year.  The investigator must also keep the IRB informed of any significant AEs. All IRB 
approved documents as well as relevant study correspondence should be copied and sent to 
the UAB Central Unit. 
For this study, clinical sites will have the opportunity to participate in a Centralized IRB process 
supported by the UAB Institutional Review Board and the UAB Central Unit.  Participating sites 
will be provided with detailed instructions on required responsibilities for participating in an 
OHRP compliant Centralized IRB. 
 

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12 
CLINICAL MONITORING STRUCTURE 
12.1 Site Monitoring Plan 
Site Monitoring will be conducted to ensure adequate human subjects protection and correct 
handling/shipping of laboratory specimens.  NIAID/DMID, which has contracted for the conduct 
of this study, or its designee will conduct the site monitoring visits.  A Clinical Monitoring Plan 
will be developed to include specifics about monitoring, including a description of the number 
and type of subjects monitored, the frequency of visits, the tasks to be completed during the 
visits, and the different types of visits that will be used during the monitoring.  The monitoring 
plan will be risk based. 
 
Site visits typically may be made at standard defined intervals.  More frequent visits may be 
made if needed.  Remote site monitoring may also occur.  Monitoring visits may include, but are 
not limited to; review of the regulatory file, informed consent forms, case report forms, and 
protocol compliance.  Study monitors will meet with investigators to discuss any problems and 
actions to be taken and will document visit finding and discussions. 
 
Every effort will be made to maintain the anonymity and confidentiality of subjects during this 
study.  However, because of the experimental nature of this sampling study, the investigator 
agrees to allow representatives of the NIAID/DMID, or the UAB Central Unit to inspect the 
facilities used in this study and to inspect, for purposes of verification, the hospital, clinic or 
study records of all subjects enrolled. 
 
As outlined in the clinical monitoring plan for this study, the  study monitors will verify that the 
subjects are consented and the clinical trial is conducted in compliance with the protocol, ICH 
E6(R1), and the applicable regulatory requirements.  Reports will be submitted to DMID and 
UAB on monitoring activities.  The study site research team will provide direct access to all trial-
related sites, source data/documents, and reports for the purpose of monitoring and auditing by 
the sponsor and their designees. 

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13 
DATA HANDLING AND RECORD KEEPING   
The investigator is responsible to ensure the accuracy, completeness, legibility, and timeliness 
of the data reported. 
Electronic case report forms (eCRFs) will be developed by the UAB Statistical and Data 
Coordinating Center.  The eCRFs will be provided electronically by the UAB Central Unit.  
Original data will be recorded on source documents (e.g., medical records, research progress 
notes, Source Document Worksheets documenting research related procedures).  The DMID 
11-0070 Data Management Plan provides a description of how the system will function.  Source 
Document Worksheets that mirror each data field on the eCRF will be available for use by sites 
as a tool to record and maintain data for each study participant enrolled in the study when other 
source documents are not used to collect original data.  All source documents should be 
completed in a neat, legible manner to ensure accurate interpretation of data.  When making a 
change or correction, 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.  Specific guidance to investigators and study staff on making corrections to source 
documents and eCRFs will be provided in the MOP for this study. 
Data recorded on the eCRF that differ from source documents must be explained on the 
Comments eCRF and in the subject’s source documents. 
13.1 
Data Management Responsibilities 
All eCRFs must be reviewed by the investigator’s research team, under the supervision of the 
investigator, who will ensure that they are accurate and complete.  All data must be supported 
by source documents, which will remain available for review by regulatory personnel and 
monitors.  Adverse events must be graded, assessed for intensity and causality, and reviewed 
by the site investigator or designee. 
Data collection is the responsibility of the clinical trial staff at the site under the supervision of 
the site PI.  During the study, the investigator must maintain complete and accurate 
documentation for the study. 
The UAB Central Unit and Statistical and Data Coordinating will be responsible for data 
management, quality review, analysis of the study data, and writing of the clinical study report.  
These tasks are detailed in the DMID 11-0070 DMP. 
13.2 
Data Capture Methods 
Clinical and laboratory data will be entered into a 21 CRF Part 11 compliant electronic Data 
Entry System (eDES)  provided by the UAB Statistical and Data Coordinating Center.  The data 

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system includes password protection and internal quality checks, such as automated range 
checks, to identify data that appear to be inconsistent, incomplete, or inaccurate. 
13.3 
Types of Data 
Data for this study will include clinical laboratory and virologic results, and clinical and outcome 
measures (e.g., virology, demographics). 
13.4 
Timing/Reports 
There are no planned interim analyses or safety reviews for this trial.  There also are no 
stopping rules. 
13.5 
Study Records Retention 
Records and documents pertaining to the conduct of this study, including source documents 
and consent forms, must be retained by the investigator for at least 2 years following completion 
of the study.  No study records shall be destroyed without prior authorization from the UAB 
Central Unit and NIAID/DMID.  These documents should be retained for a longer period, 
however, if required by local regulations.  It is the responsibility of the sponsor to notify the UAB 
Central Unit, which will notify the investigators, when these documents no longer need to be 
retained. 
13.6 
Protocol Deviations 
Each investigator must adhere to the protocol as detailed in this document and agree that any 
changes to the protocol must be approved by the UAB Central Unit and NIAID/DMID prior to 
seeking approval from the IRB/IEC.  Each investigator will be responsible for enrolling only 
those study participants who have met protocol eligibility criteria. 
A protocol deviation is any noncompliance with the clinical trial protocol, GCP, or Manual of 
Procedures requirements.  The noncompliance may be either on the part of the study 
participant, 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 GCP sections: 
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 to use continuous vigilance to identify and report deviations 
within 1 business day of identification of the protocol deviation that increases subject risk.  

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Deviations that do not increase subject risk can be reported within 5 business days of 
knowledge of the event.  All deviations must be promptly reported to the UAB Central Unit.  UAB 
will report all deviations to DMID in accordance with DMID’s instructions. 
All deviations from the protocol must be addressed in the source documents.  A completed copy 
of the DMID protocol deviation form must be maintained in the regulatory file (Project Notebook 
or designated location) as well as in the subject’s source documents.  A log of protocol deviation 
will be maintained in the Project Notebook.  Protocol deviations must be sent to the local IRB 
per the IRB’s guidelines.  The site PI/study staff is responsible for knowing and adhering to their 
IRB/IEC requirements. 

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14 
PUBLICATION POLICY 
Following completion of this study, the investigators are expected to publish the results in a 
scientific journal.  All research reports and other publications resulting from the work completed 
in this protocol shall: 
• 
Acknowledge the support of the National Institutes of Health whenever publicizing the 
results from this clinical trial in any media. 
• 
Be submitted to the Project Director in the form of advance copies for review and 
comment prior to the publication to ensure appropriate coordination of the research 
results. 
• 
Be furnished in a list of publications resulting from the research as part of the annual 
progress report submitted to the principal investigator. 
The International Committee of Medical Journal Editors (ICMJE) member journals has 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, which is sponsored by the 
National Library of Medicine.  Other biomedical journals are considering adopting similar 
policies. 
Unless exempted, this trial will be registered prior to enrollment of study subjects.  It is the 
responsibility of the study’s PI (e.g., Dr. Kimberlin) to register the non-exempted trials and post 
results in compliance with Public Law 110-85, the Food and Drug Administration Amendments 
Act of 2007 (FDAAA). 

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15 
LITERATURE REFERENCES 
1. 
Brown ZA, Wald A, Morrow RA, et al. Effect of serologic status and cesarean delivery on 
transmission rates of herpes simplex virus from mother to infant. JAMA. 2003;289:203-9. 
2. 
Whitley RJ, Nahmias AJ, Soong SJ, et al. Vidarabine therapy of neonatal herpes simplex 
virus infection. Pediatrics. 1980;66:495-501. 
3. 
Whitley RJ, Yeager A, Kartus P, et al. Neonatal herpes simplex virus infection: follow-up 
evaluation of vidarabine therapy. Pediatrics. 1983;72:778-85. 
4. 
Whitley R, Arvin A, Prober C, et al. A controlled trial comparing vidarabine with acyclovir 
in neonatal herpes simplex virus infection. N Engl J Med. 1991;324:444-9. 
5. 
Kimberlin DW, Lin CY, Jacobs RF, et al. Safety and efficacy of high-dose intravenous 
acyclovir in the management of neonatal herpes simplex virus infections. Pediatrics. 
2001;108:230-8. 
6. 
Whitley RJ, Nahmias AJ, Visintine AM, et al. The natural history of herpes simplex virus 
infection of mother and newborn. Pediatrics. 1980;66:489-94. 
7. 
Kimberlin DW, Whitley RJ, Wan W, et al. Oral acyclovir suppression and 
neurodevelopment after neonatal herpes. N Engl J Med. 2011;365:1284-1292. 
8. 
Kimberlin DW. Neonatal herpes simplex infection. Clin Microbiol Rev. 2004;17:1-13. 
9. 
Whitley RJ, Corey L, Arvin A, et al. Changing presentation of herpes simplex virus 
infection in neonates. J Infect Dis. 1988;158:109-16. 
10. 
Stanberry LR, Cunningham AL, Mindel A, et al. Prospects for control of herpes simplex 
virus disease through immunization. Clinical Infectious Diseases. 2000;30:549-66. 
11. 
Kimberlin DW, Rouse DJ. Genital herpes. N Engl J Med. 2004;350:1970-7. 
12. 
Stanberry LR, Spruance SL, Cunningham AL, et al. Glycoprotein-D-adjuvant vaccine to 
prevent genital herpes. New England Journal of Medicine. 2002;347:1652-61. 
13. 
Belshe RB, Leone PA, Bernstein DI, et al. Efficacy results of a trial of a herpes simplex 
vaccine. N Engl J Med. 2012;366:34-43. 
14. 
Xu F, Sternberg MR, Kottiri BJ, et al. Trends in herpes simplex virus type 1 and type 2 
seroprevalence in the United States. JAMA. 2006;296:964-973. 
15. 
Yeager AS, Arvin AM. Reasons for the absence of a history of recurrent genital 
infections in mothers of neonates infected with herpes simplex virus. Pediatrics. 
1984;73:188-93. 
16. 
Braig S, Luton D, Sibony O, et al. Acyclovir prophylaxis in late pregnancy prevents 
recurrent genital herpes and viral shedding. Eur J Obstet Gynecol Reprod Biol. 
2001;96:55-8. 
17. 
Scott LL, Sanchez PJ, Jackson GL, et al. Acyclovir suppression to prevent cesarean 
delivery after first-episode genital herpes. Obstet Gynecol. 1996;87:69-73. 
18. 
Scott LL, Hollier LM, McIntire D, et al. Acyclovir suppression to prevent recurrent genital 
herpes at delivery. Infect Dis Obstet Gynecol. 2002;10:71-7. 
19. 
Sheffield JS, Hill JB, Hollier LM, et al. Valacyclovir Prophylaxis to Prevent Recurrent 
Herpes at Delivery: A Randomized Clinical Trial. Obstet Gynecol. 2006;108:141-147. 
20. 
Brocklehurst P, Kinghorn G, Carney O, et al. A randomised placebo controlled trial of 
suppressive acyclovir in late pregnancy in women with recurrent genital herpes infection. 
British Journal of Obstetrics & Gynaecology. 1998;105:275-80. 
21. 
Andrews WW, Kimberlin DF, Whitley R, et al. Valacyclovir therapy to reduce recurrent 
genital herpes in pregnant women. Am J Obstet Gynecol. 2006;194:774-781. 

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22. 
Pinninti SG, Feja KN, Kimberlin DW, et al. Neonatal herpes disease despite maternal 
antenatal antiviral suppressive therapy: A multicenter case series of the first such infants 
reported., 48th Annual Meeting of The Infectious Diseases Society of America (IDSA), 
Vancouver, British Columbia; Abstract #1841, October 22, 2010 
23. 
Pinninti SG, Angara R, Feja KN, et al. Neonatal herpes disease following maternal 
antenatal antiviral suppressive therapy: a multicenter case series. J Pediatr. 
2012;161:134-8 e1-3. 
24. 
Brown ZA, Benedetti J, Ashley R, et al. Neonatal herpes simplex virus infection in 
relation to asymptomatic maternal infection at the time of labor. N Engl J Med. 
1991;324:1247-52. 
25. 
Brown ZA, Vontver LA, Benedetti J, et al. Effects on infants of a first episode of genital 
herpes during pregnancy. N Engl J Med. 1987;317:1246-51. 
26. 
Nahmias AJ, Josey WE, Naib ZM, et al. Perinatal risk associated with maternal genital 
herpes simplex virus infection. Am J Obstet Gynecol. 1971;110:825-37. 
27. 
Gardella C, Huang ML, Wald A, et al. Rapid polymerase chain reaction assay to detect 
herpes simplex virus in the genital tract of women in labor. Obstet Gynecol. 
2010;115:1209-16. 
28. 
Regan JA, Klebanoff MA, Nugent RP. The epidemiology of group B streptococcal 
colonization in pregnancy. Vaginal Infections and Prematurity Study Group. Obstet 
Gynecol. 1991;77:604-10. 
29. 
Yancey MK, Schuchat A, Brown LK, et al. The accuracy of late antenatal screening 
cultures in predicting genital group B streptococcal colonization at delivery. Obstet 
Gynecol. 1996;88:811-5. 
30. 
Campbell JR, Hillier SL, Krohn MA, et al. Group B streptococcal colonization and 
serotype-specific immunity in pregnant women at delivery. Obstet Gynecol. 
2000;96:498-503. 
31. 
Centers for Disease Control and Prevention. Prevention of Perinatal Group B 
Streptococcal Disease: Revised Guidelines from CDC, 2010. MMWR Morb Mortal Wkly 
Rep. 2010;59:1-32. 
32. 
Cheng PJ, Chueh HY, Liu CM, et al. Risk factors for recurrence of group B 
streptococcus colonization in a subsequent pregnancy. Obstet Gynecol. 2008;111:704-
9. 
33. 
Turrentine MA, Ramirez MM. Recurrence of group B streptococci colonization in 
subsequent pregnancy. Obstet Gynecol. 2008;112:259-64. 
34. 
Wald A, Corey L, Cone R, et al. Frequent genital herpes simplex virus 2 shedding in 
immunocompetent women. Effect of acyclovir treatment. Journal of Clinical Investigation. 
1997;99:1092-7. 
35. 
Boyer KM, Gadzala CA, Kelly PD, et al. Selective intrapartum chemoprophylaxis of 
neonatal group B streptococcal early-onset disease. II. Predictive value of prenatal 
cultures. J Infect Dis. 1983;148:802-9. 
36. 
Arvin AM, Hensleigh PA, Prober CG, et al. Failure of antepartum maternal cultures to 
predict the infant's risk of exposure to herpes simplex virus at delivery. N Engl J Med. 
1986;315:796-800. 
37. 
Mark KE, Wald A, Magaret AS, et al. Rapidly cleared episodes of oral and anogenital 
herpes simplex virus shedding in HIV-infected adults. J Acquir Immune Defic Syndr. 
2010;54:482-8. 

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38. 
Schiffer JT, Abu-Raddad L, Mark KE, et al. Mucosal host immune response predicts the 
severity and duration of herpes simplex virus-2 genital tract shedding episodes. Proc 
Natl Acad Sci U S A. 2010;107:18973-8. 
 
 
 

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SUPPLEMENTS/APPENDICES 

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APPENDIX A: SCHEDULE OF EVENTS 
 
Table 1.  Schematic of Study Design 
 
Study Day 
 
≤ 1 
1 
5 
10 
20 
30 
60 
90 
Nonpregnant Women in STI Clinics (Group 1) 
 
 
 
 
 
 
 
 
Verification that inclusion/exclusion criteria are met, and 
informed consent is obtained 
X 
 
 
 
 
 
 
 
Lesion swab from women 
 
Xa,b 
 
 
 
 
 
 
Collect whole blood for type-specific serology from women (at 
least 0.5 mL of serum required) 
 
Xc 
 
 
 
 
 
 
Pregnant Women Admitted with the Intent of Delivery 
(Group 2) 
 
 
 
 
 
 
 
 
Verification that inclusion/exclusion criteria are met, and 
informed consent is obtained 
X 
 
 
 
 
 
 
 
Vaginal swab from women 
 
Xd 
 
 
 
 
 
 
Collect whole blood for type-specific serology from women (at 
least 0.5 mL of serum required) 
 
Xe 
 
 
 
 
 
 
Provide educational fact sheet about neonatal herpes 
 
Xf 
 
 
 
 
 
 
Follow-up contact re: treatment for neonatal HSV and maternal 
mode of delivery 
 
 
 
 
 
Xg 
 
a. Lesion swabs from nonpregnant women will be placed in viral transport media, refrigerated at 2-8°C, 
and sent to the laboratory for real time analysis by HSV culture on a portion of the transport media, 
and the remainder of the transport media will be frozen in aliquots for subsequent batch testing by 
routine HSV PCR and Xpert HSV 1/2 PCR at the UAB Central Laboratory 
b. HSV culture results from women in Group 1 will be reported to their treating physician 
c. Blood for serologic testing will be collected from all nonpregnant women enrolled, but testing will be 
performed only on samples from those women who are determined to be shedding HSV genitally by 
culture, routine PCR, or Xpert HSV 1/2 PCR 
d. Vaginal swabs from pregnant women will be placed in viral transport media, frozen at -20°C or colder, 
and batch-shipped to the UAB Central Laboratory for batched analysis as follows:  specimens from 
approximately half of the women in Group 2 will be tested by routine HSV PCR and Xpert HSV 1/2 
PCR; specimens from the rest of the women in Group 2 will be stored for possible testing in the future 
by routine HSV PCR and Xpert HSV 1/2 PCR. 
e. Blood for serologic testing will be collected (or remnant sample may be retrieved) from all pregnant 
women enrolled (window: ± 3 days), but testing will be performed only on samples from pregnant 
women who are determined to be shedding HSV genitally by routine HSV PCR or Xpert HSV 1/2 
PCR 
f. 
Printed material about neonatal HSV signs and symptoms will be provided to all pregnant women 
enrolling in the study 
g. Follow-up contact will involve all Group 2 women enrolled on the study, or their designated 
representative.  Inquiry will be made regarding information suggesting development of neonatal HSV 
disease in the first 4-6 weeks of life.  Maternal receipt of antiviral therapy at any point following 
enrollment will be captured.