Prospective Cohort Study of HIV and Zika in Infants and Pregnancy
Conditions: HIV, Zika Virus, Pregnant Women
Sponsor: Westat

HIV ZIP 
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Version 1.0  
March 20, 2017 
Prospective Cohort Study of HIV and Zika in Infants and Pregnancy  
(HIV ZIP) 
Sponsored by: 
The Eunice Kennedy Shriver 
National Institute of Child Health and Human Development (NICHD) 
Protocol Co-Chairs: 
George R. Seage III, DSc, MPH  
 
Marisa M. Mussi-Pinhata, MD 
 
Protocol Vice-Chair: 
Ann Aschengrau, ScD 
 
NICHD Medical Officers: 
Nahida Chakhtoura, MD, MsGH 
 
John Moye, Jr., MD 
 
NICHD Program Officer: 
Rohan Hazra, MD 
 
NIAID Investigator:  
Walla Dempsey, PhD 
 
Protocol Specialist: 
Dina Monte, RN, BSN, CCRC 
 
 

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Table of Contents 
Chapter 
Page 
 
Protocol Team Roster ............................................................................  
7 
 
Signature Page .......................................................................................  
9 
 
Study Management ................................................................................  
10 
 
List of Abbreviations .............................................................................  
11 
 
Study Abstract ........................................................................................  
14 
 
Maternal Study Schema .........................................................................  
19 
 
Maternal Study Schema (ZIKV-Symptomatic or Asymptomatic 
with Positive Serology) ..........................................................................  
20 
 
Infant Study Schema ..............................................................................  
21 
1.0 
Introduction ............................................................................................  
22 
1.1 
Background on ZIKV ................................................................  
22 
1.2 
Background on HIV Infection in Pregnant Women ..................  
25 
1.3 
Rationale for HIV ZIP Study .....................................................  
29 
2.0 
Study Objectives ....................................................................................  
30 
2.1 
Primary Objectives.....................................................................  
30 
2.2 
Secondary Objectives.................................................................  
30 
3.0 
Study Design ..........................................................................................  
31 
3.1 
Study Population ........................................................................  
31 
3.2 
Sample Size ................................................................................  
32 
4.0 
Protocol Registration Procedures ...........................................................  
32 
5.0 
Selection and Enrollment of Study Participants ....................................  
32 
5.1 
Maternal Inclusion Criteria ........................................................  
32 
5.2 
Maternal Exclusion Criteria .......................................................  
33 
5.3 
Infant Inclusion Criteria .............................................................  
33 
 
 

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Contents (continued) 
Chapter 
Page 
5.4 
Infant Exclusion Criteria ............................................................  
34 
5.5 
Participant Recruitment .............................................................  
34 
5.6 
Informed Consent.......................................................................  
34 
5.7 
Co-enrollment Guidelines ..........................................................  
35 
6.0 
Study Evaluations ..................................................................................  
35 
6.1 
Maternal Evaluations .................................................................  
35 
6.2 
Infant Evaluations ......................................................................  
38 
7.0 
Study Schedule and Procedures .............................................................  
40 
7.1 
Maternal Study Visits ................................................................  
40 
7.2 
Infant Study Visits .....................................................................  
47 
8.0 
Specimen Tracking and Storage ............................................................  
49 
8.1 
Specimen Tracking ....................................................................  
49 
8.2 
Biorepository Storage ................................................................  
49 
9.0 
Participant and Study Management .......................................................  
50 
9.1 
Tracking Participants / Follow-up .............................................  
50 
9.2 
Retention ....................................................................................  
50 
9.3 
Study Visit Management............................................................  
50 
9.4 
Premature Study Discontinuation ..............................................  
50 
9.5 
Death of a Participant.................................................................  
51 
10.0 
Data Collection and Management ..........................................................  
51 
10.1 
Development of Protocol and Case Report Forms ....................  
51 
10.2 
Data Records ..............................................................................  
51 
10.3 
Data Submission ........................................................................  
52 
10.4 
Data Quality Assurance .............................................................  
52 
10.5 
Clinical Site Monitoring and Record Availability .....................  
52 
10.6 
Retention of Clinical Research Records ....................................  
53 
10.7 
Protocol Query Management .....................................................  
53 
10.8 
Protocol Deviations ....................................................................  
53 
10.9 
Collaboration with Outside Studies ...........................................  
54 
 
 

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Contents (continued) 
Chapter 
Page 
11.0 
Monitoring Unanticipated Problems ......................................................  
54 
11.1 
Unanticipated Problems Definition............................................  
55 
11.2 
Unanticipated Problems Categories ...........................................  
55 
11.3 
Reporting Requirements ............................................................  
56 
11.4 
Grading of Impact ......................................................................  
56 
12.0 
Data Analysis Plan .................................................................................  
57 
12.1 
Aim 1: To Determine the Feasibility of Enrolling 
Pregnant Women with ZIKV/HIV Co-infection, HIV 
Infection Alone, ZIKV Infection Alone as well as 
Doubly Uninfected Women into a Prospective Cohort 
Study at Selected Sites in the Continental U.S., P.R. and 
Brazil ..........................................................................................  
57 
12.2 
Aim 2: To Compare HIV Viral Suppression in HIV-
Infected Women with and without ZIKV Co-infection 
during Pregnancy and the Time of Delivery ..............................  
57 
12.3 
Aim 3: To Compare the Incidence of ZIKV Infection 
among Pregnant Women with HIV Infection and Those 
without HIV Infection ................................................................  
57 
12.4 
Aim 4: To Compare the Incidence of Adverse 
Pregnancy Outcomes between Women Co-infected 
with HIV and ZIKV, Women Infected with Either HIV 
or ZIKV Alone and Doubly Uninfected Women .......................  
58 
12.5 
Aim 5: To Compare the Incidence of Vertical 
Transmission of HIV and ZIKV between Women Co-
infected with HIV and ZIKV, and Women Infected 
with Either HIV or ZIKV Alone ................................................  
58 
12.6 
Aim 6: To Compare the Incidence of Congenital 
Malformations and Other Adverse Outcomes (Including 
Microcephaly, Neonatal Death, CNS Malformations, 
Hydrops and Ocular Abnormalities) among Offspring 
of Women Co-infected with HIV and ZIKV, Women 
Infected with Either HIV or ZIKV Alone and Doubly 
Uninfected Women ....................................................................  
58 
12.7 
Aim 7: To Compare Long-term Growth, Hearing, 
Vision and Neurodevelopment Outcomes in Children 
with In Utero Exposure to HIV and ZIKV, In Utero 
Exposure to Either HIV or ZIKV Alone and No In 
Utero Exposure to Either Virus..................................................  
59 

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Contents (continued) 
Chapter 
Page 
12.8 
Missing, Unused and Spurious Data ..........................................  
59 
12.9  Power and Sample Size ..............................................................  
59 
12.10 Interim Monitoring.....................................................................  
64 
13.0 
Human Subjects .....................................................................................  
65 
13.1 
45 CFR § 46.404 ........................................................................  
65 
13.2 
Institutional Review Board / Institutional Ethics 
Committee and Informed Consent .............................................  
65 
13.3 
Prisoner Participation .................................................................  
65 
13.4 
Participant Confidentiality .........................................................  
66 
13.5 
Risks and Benefits......................................................................  
66 
13.6 
Biorepository Policies ................................................................  
67 
13.7 
45 CFR § 160 and 164 Standards for Privacy of 
Individually Identifiable Health Information (“Privacy 
Rule” Pursuant to the HIPAA) ...................................................  
67 
13.8 
Study Discontinuation ................................................................  
68 
14.0 
Publication of Research Findings ..........................................................  
68 
15.0 
Biohazard Containment .........................................................................  
68 
16.0 
References ..............................................................................................  
69 
Appendices 
I 
Maternal Schedule of Evaluations for Asymptomatic and 
Symptomatic Women.............................................................................  
77 
II 
Infant Schedule of Evaluations ..............................................................  
79 
III 
List of Participating Sites and Investigators ..........................................  
81 
IV 
Sample Maternal Informed Consent ......................................................  
83 
V 
Sample Consent for Infant Participation ................................................  
83 
 
 

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Contents (continued) 
Tables 
Page 
12-1. 
Precision of event rate estimates for various levels of accrual, 
overall or within subgroups defined by HIV status ...............................  
60 
12-2. 
Minimum detectable ORs (and percent with unsuppressed VL 
among ZIKV-infected) at 80% power comparing HIV+/ZIKV+ 
vs. HIV+/ZIKV- women ........................................................................  
61 
12-3. 
Minimum detectable ORs (and percent of ZIKV-infected 
among HIV-infected) at 80% power for various sample sizes 
and assumed ZIKV infection rates among HIV-uninfected ..................  
62 
12-4. 
Power for detecting differences in rate of adverse pregnancy 
outcomes (for Aims 4 and 6) based on assumed proportion in 
the HIV and ZIKV co-infected, HIV-infected and ZIKV-
infected groups as compared to the HIV and ZIKV-uninfected 
group ......................................................................................................  
63 
 
 

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Protocol Team Roster 
Protocol Co-Chair 
George R. Seage III, DSc, MPH 
Harvard T. H. Chan School of Public Health 
Department of Epidemiology 
677 Huntington Avenue 
Kresge Building Room 816A 
Boston, MA 02115 
Phone: 617-432-4567 
Email: gseage@hsph.harvard.edu 
 
Protocol Co-Chair 
Marisa M. Mussi-Pinhata, MD 
Department of Pediatrics 
Ribeirão Preto Medical School 
University of Sao Paulo 
Avenida Bandeirantes 3900 
Ribeirão Preto 
Sao Paulo, 14049-900, Brazil 
Phone: 55-16-3602-2807 
Email: mmmpinha@fmrp.usp.br 
 
NICHD Medical Officer  
Nahida Chakhtoura, MD, MsGH 
MPIDB/NICHD/NIH 
6710 Rockledge Drive, Room 2155D, MSC 
7002 
Bethesda, MD 20817 
Phone: 301-435-6872 
Email: nahida.chakhtoura@nih.gov 
 
NICHD Medical Officer 
John Moye, Jr., MD  
MPIDB/NICHD/NIH 
6710B Rockledge Drive, Room 2146, MSC 
7002 
Bethesda, MD 20817 
Phone: 301-594-8624  
Email: john.moye@nih.hhs.gov 
 
Protocol Vice-Chair  
Ann Aschengrau, ScD 
Boston University School of Public Health  
Department of Epidemiology  
715 Albany Street Talbot  
Boston, MA 02118  
Phone: 617-638-5228 
Email: aaschen@bu.edu  
 
Protocol Specialist 
Dina Monte, RN, BSN, CCRC 
Westat 
1600 Research Boulevard, WB420C 
Rockville, MD 20850 
Phone: 914-591-6170 
Email: dinamonte@westat.com 
 
Protocol Epidemiologist 
Kunjal Patel, DSc, MPH 
Harvard T. H. Chan School of Public Health 
Department of Epidemiology  
677 Huntington Avenue 
Kresge 816C 
Boston, MA 02115 
Phone: 617-432-3174 
Email: kpatel@hsph.harvard.edu 
 
Protocol Statistician 
Paige L. Williams, PhD 
Harvard T. H. Chan School of Public Health  
Departments of Biostatistics and Epidemiology 
665 Huntington Avenue 
Building I, Room 415 
Boston, MA 02115 
Phone: 617-432-3872 
Email: paige@hsph.harvard.edu  
 

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Protocol Team Roster (continued) 
Protocol Laboratory Specialist  
Frances Whalen, MPH, MT (ASCP) 
Westat 
1600 Research Boulevard  
Rockville, MD 20850 
Phone: 704-422-4066 
Email: franceswhalen@westat.com 
NICHD Program Officer 
Rohan Hazra, MD 
Branch Chief 
MPIDB/NICHD/NIH 
6710B Rockledge Drive 
Room 2113, MSC 7002 
Rockville, MD 20817 
Phone: 301-435-6868 
Email: rohan.hazra@nih.gov 
 
Protocol Clinical Data Manager 
Donna Butler, RN, BSN 
Westat 
1600 Research Boulevard 
Rockville, MD 20850 
Phone: 301-738-3680 
Email: donnabutler@westat.com 
 
NIAID Investigator 
Walla Dempsey, PhD 
Program Officer for Clinical Research 
Division of Microbiology and Infectious 
Diseases, Virology Branch 
NIAID/NIH 
5601 Fishers Lane 
Room 8E57, MSC 9825 
Bethesda, MD  20892-9825 
Phone: 240-292-4197  
Email: wdempsey@niaid.nih.gov 
 
 
 
 
 

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Signature Page 
I, the Principal Investigator, agree to conduct this study in full accordance with the provisions of 
this protocol and will ensure that all associates, colleagues, and employees assisting in the 
conduct of the study are informed about the obligations incurred by their contributions to the 
study. 
 
My signature below constitutes approval of this protocol (Prospective Cohort Study of HIV and 
Zika in Infants and Pregnancy (HIV ZIP) and the attachments and provides the necessary 
assurances that this study 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 United States (U.S.) federal regulations and International 
Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals 
for Human Use (ICH) guidelines. It is understood that no deviations from the protocol may be 
made without permission of the Sponsor.  
 
 
Site Name: _______________________________________________ 
 
Site Principal Investigator:* 
 
Signed: 
 
Date:  
 
Name 
Title 
 
 
 
* The protocol should be signed by the local investigator of record who is responsible for the 
study implementation at his or her specific site.  
 
 

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Study Management 
Before the recruitment and enrollment of participants, participating research sites must have the 
protocol and consent forms approved by their local Institutional Review Boards 
(IRBs)/Institutional Ethics Committees (IECs). In addition, research sites must receive protocol 
registration approval from the Regulatory Associate at Westat. All original approved documents 
must be maintained at the research site. A detailed description of site and protocol registration 
procedures is included in the HIV ZIP Manual of Procedures (MOP).  
 
All queries for this protocol should be sent to the HIV ZIP Protocol Team using the HIV ZIP 
Query and Notification System (QNS) accessible via the HIV ZIP page on the NICHD Clinical 
Studies website (www.nichdclinicalstudies.org). The appropriate team member will respond to 
queries generally within 48 business hours via the HIV ZIP QNS and copy the other team 
members. The Protocol Manager, with the help of other Westat personnel and/or NICHD, if 
necessary, will answer general protocol implementation, case report form (CRF) completion, and 
specimen shipping queries. The Protocol Co-Chairs or their designees will respond to eligibility, 
study and participant management, exemptions and/or adverse event (AE) queries and 
notifications.  
 
Queries and replies will automatically be archived at Westat. The Protocol Manager will post 
those queries deemed relevant to all sites on the NICHD Clinical Studies/HIV ZIP website, 
where they will be available for future reference to the protocol team and participating sites. 
 
For protocol registration questions, email Regulatory@westat.com or call 1-888-464-5246 (toll-
free) or 00 1 240-453-5661 (long distance charges). Protocol registration material can be sent 
electronically to Regulatory@westat.com or via fax at 1-888-865-1983 or 00 1 240-455-5580 
(long distance charges).  
 
 

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List of Abbreviations 
AABR 
Automated Auditory Brainstem Response 
Ab 
Antibody 
AE 
Adverse Event 
Ag 
Antigen 
AIDS 
Acquired Immunodeficiency Syndrome 
ArboNET 
National Arbovirus Surveillance System 
ART 
Antiretroviral Therapy 
ARV 
Antiretroviral  
ASQ 
Ages and Stages Questionnaire 
βhCG 
Beta Human Chorionic Gonadotropin 
BSID 
Bayley Scales of Infant and Toddler Development 
cART 
Combination Antiretroviral Therapy 
CDC 
Centers for Disease Control and Prevention 
CFR 
Code of Federal Regulations 
CI 
Confidence Interval 
CKNV 
Chikungunya Virus 
CMV 
Cytomegalovirus 
CNS 
Central Nervous System 
CRF 
Case Report Form 
CSF 
Cerebrospinal Fluid 
CT 
Computerized Tomography 
DAIDS 
Division of AIDS 
DENV 
Dengue Virus 
ELISA 
Enzyme-Linked Immunosorbent Assay 
FDA 
Food and Drug Administration 
GA 
Gestational Age 
GBS 
Guillain-Barré Syndrome 
GCP 
Good Clinical Practice 
HAART 
Highly Active ART 
HBV 
Hepatitis B Virus 
HCV 
Hepatitis C Virus 

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List of Abbreviations (continued) 
HEU 
HIV-Exposed Uninfected 
HHS 
The U.S. Department of) Health and Human Services 
HINE 
Hammersmith Infant Neurological Examination 
HIPAA 
Health Insurance Portability and Accountability Act 
HIV 
Human Immunodeficiency Virus 
HIV ZIP 
Prospective Cohort Study of HIV and Zika in Infants and 
Pregnancy 
HPTN 
HIV Prevention Trials Network 
IATA 
International Air Transport Association 
IC 
Informed Consent 
ICH 
International Conference on Harmonisation of Technical 
Requirements for Registration of Pharmaceuticals for Human 
Use 
IEC 
Independent or Institutional Ethics Committee 
IgM 
Immunoglobulin M 
IRB 
Institutional Review Board 
LDMS 
Laboratory Data Management System 
LMP 
Last Menstrual Period 
LPC 
Laboratory Processing Chart 
MAC-ELISA 
IgM Ab Capture ELISA 
MOP 
Manual of Procedures 
MOU 
Memorandum of Understanding 
MPIDB 
Maternal and Pediatric Infectious Disease Branch 
MRI 
Magnetic Resonance Imaging 
MTCT 
Mother-to-Child Transmission 
ND 
Neurodevelopmental 
NIAID 
National Institute of Allergy and Infectious Diseases 
NICHD 
National Institute of Child Health and Human Development 
NIH 
National Institutes of Health 
NNRTI 
Non-Nucleoside Reverse Transcriptase Inhibitor 
NRTI 
Nucleoside Reverse Transcriptase Inhibitor 
OAE 
Otoacoustic Emission 

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List of Abbreviations (continued) 
OHRP 
Office for Human Research Protections 
OR 
Odds Ratio 
PACTG 
Pediatric AIDS Clinical Trials Group  
PAHO 
Pan American Health Organization 
PCR 
Polymerase Chain Reaction  
PE 
Physical Examination 
PHACS 
Pediatric HIV/AIDS Cohort Study 
PI 
Principal Investigator 
PID 
Participant Identification 
PII 
Personally Identifying Information  
P.R. 
Puerto Rico 
PRNT 
Plaque Reduction Neutralization 
QNS 
Query and Notification System 
RDC 
Remote Data Capture 
RNA 
Ribonucleic Acid 
RPR 
Rapid Plasma Reagin 
RT-PCR 
Reverse Transcription PCR  
SID 
Screening Identification  
SMARTT 
Surveillance Monitoring for Antiretroviral Therapy Toxicities 
TORCH 
Toxoplasmosis, Rubella, CMV, Herpes 
UNAIDS 
The Joint United Nations Programme on HIV/AIDS 
US 
Ultrasound 
U.S. 
United States 
VL 
Viral Load 
VS 
Vital Signs 
WHO 
World Health Organization 
WNV 
West Nile Virus 
YFV 
Yellow Fever Virus 
ZDV 
Zidovudine 
ZIKV 
Zika Virus 
ZIP 
International Prospective Observational Cohort Study of Zika in 
Infants and Pregnancy 

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Study Abstract 
Design: 
This is a two-phase prospective international cohort study of pregnant women 
and their infants from those pregnancies whose goals are to compare the 
incidence of Zika virus (ZIKV) infection among pregnant women with and 
without Human Immunodeficiency Virus (HIV) infection and to determine the 
risk of adverse maternal and child outcomes associated with ZIKV/HIV co-
infection across clinical sites in the continental United States (U.S.), Puerto Rico 
(P.R.) and Brazil.  
Phase I will enroll pregnant women/infant pairs who are: (1) infected with HIV 
only; (2) infected with ZIKV only; (3) infected with HIV and ZIKV; and (4) not 
infected with HIV or ZIKV. Phase I will assess the feasibility of enrolling a total 
of 200 pregnant women/infant pairs within a year, with a target of 150 HIV-
infected women, 50 HIV-uninfected women from the continental U.S. sites only, 
and a minimum of 20 who are co-infected with HIV and ZIKV.  
Should the feasibility of Phase I prove successful, Phase II will commence by 
enrolling up to 1,800 additional pregnant women/infant pairs to the 4 groups 
described above. The comparison group of HIV-uninfected pregnant 
women/infant pairs from P.R and Brazil (ZIKV-infected and uninfected) will be 
obtained from data collected in the concurrent International Prospective 
Observational Cohort Study of Zika in Infants and Pregnancy (ZIP study). 
All HIV-infected and uninfected study participants will be tested for ZIKV. 
Enrolled women will be followed throughout their pregnancy and up to six 
weeks postpartum. Infants born to enrolled women will be followed for a full 
year after birth. Thereafter, the infants born to women at continental U.S. and 
P.R. sites also implementing the Surveillance Monitoring for Antiretroviral 
Therapy (ART) Toxicities (SMARTT) study may be followed up yearly until 
adulthood through SMARTT depending on the availability of funds. The data 
analysis will be conducted to address the objectives listed below.  
Sample size: 
Phase I: A total of 200 HIV-infected and uninfected pregnant women and their 
children (mother/infant pairs) from that pregnancy.  
Phase II: Up to 1,800 additional HIV-infected and uninfected pregnant women 
and their children from that pregnancy. 
Duration: 
Depending on the success of Phase I, the duration of the study may vary from 
four to six years. As described below, its duration will be four years if Phase I 
does not demonstrate feasibility, and six years if Phase I demonstrates 
feasibility.  
The duration of participation for pregnant women enrolled in Phase I is from the 
time of enrollment through approximately six weeks postpartum. The duration of 
participation for infants born to enrolled women is from birth through 
approximately one year of age. 
 Year 1- 200 pregnant women at risk for ZIKV and their newborn infants 
will be recruited and assessments begun.  
 Year 2- Completion of infant enrollments and maternal assessments, and 
continuation of infant assessments. 

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 Year 3- Completion of one-year follow-up of all infants. 
 Year 4- Completion of all Phase I laboratory assays, reporting of results 
and data analyses. 
At the end of Year 1, we will assess the success of Phase I. If the feasibility 
phase proves successful, enrollment into Phase II will begin. Irrespective of 
feasibility, Year 2 through 4 activities (described above) will occur for the 200 
women and their infants enrolled in Phase I. 
Phase II: The duration of participation for pregnant women and their infants is 
identical to Phase I. 
 Years 2 and 3*- Up to 1,800 pregnant women at risk for ZIKV and their 
infants will be recruited.  
 Year 4- Completion of newborn enrollments and maternal assessments, 
and continuation of infant assessments. 
 Year 5- Completion of one-year follow-up of all infants. 
 Year 6- Completion of all Phase II laboratory assays, reporting of results 
and data analyses. 
* Phase II may begin earlier than Year 2 and end earlier than Year 6 if Phase I 
enrolls 200 eligible women and proves feasible in less than one year. The 
duration of the Phase II enrollment period may be adjusted depending on the 
status of the ZIKV epidemic and availability of future funding. 
Population: 
Maternal participants:  
Pregnant women who are at risk for ZIKV are eligible for participation if they 
meet the following criteria: 
 Reside in a geographic area accessible to one of the selected clinical 
research sites; 
 Provide written informed consent (IC) (or assent with parent(s)/legal 
guardian(s) permission, where required); 
 Age 15 years and older at enrollment; 
 Confirmation of pregnancy by Beta Human Chorionic Gonadotropin 
(βhCG) measurement in blood or urine or fetal ultrasound (US) with fetal 
heart tones present; and 
 Confirmation of pregnancy at less than 18 weeks gestational age (GA) 
based on pregnancy calculator or fetal US; or  
 Confirmation of pregnancy at 18 weeks or greater GA based on pregnancy 
calculator or fetal US and with acute ZIKV symptoms and laboratory-
confirmed ZIKV infection by ZIKV ribonucleic acid (RNA) detection. 
In addition, at least one of the following three ZIKV-exposure risk categories 
must be met: 
 Has resided in for at least three months or traveled within the last three 
months to a country or U.S. Territory with active ZIKV transmission. The 
most up-to-date list of these locations can be found at 
http://www.cdc.gov/zika/geo/active-countries.html.  
 Within the U.S., the list of communities with local active ZIKV 
transmission can be found at: http://www.cdc.gov/zika/intheus/maps-zika-
us.html;  

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 Sexual partner has resided in or traveled within the last six months to a 
country or U.S. Territory with active ZIKV transmission, or was 
diagnosed with ZIKV within the previous six months; or 
 Household member has been diagnosed with ZIKV infection or has 
traveled since the woman’s last menstrual period (LMP) to a country or 
U.S. Territory with active ZIKV transmission.  
Infant participants:  
All infants born to women while enrolled in this study are eligible to participate 
if the parent(s)/legal guardian(s) provides written consent for his or her child’s 
participation. 
Primary 
objectives: 
The primary objectives of the HIV ZIP study are:  
1. To determine the feasibility of enrolling pregnant women with ZIKV/HIV 
co-infection, HIV infection alone, ZIKV infection alone as well as doubly 
uninfected women into a prospective cohort study at selected sites in the 
continental U.S., P.R. and Brazil.  
2. To compare HIV viral suppression in HIV-infected women with and 
without ZIKV co-infection during pregnancy and the time of delivery. 
3. To compare the incidence of ZIKV infection among pregnant women with 
HIV infection and those without HIV infection.  
4. To compare the incidence of adverse pregnancy outcomes between 
women co-infected with HIV and ZIKV, women infected with either HIV 
or ZIKV alone, and doubly uninfected women.  
5. To compare the incidence of vertical transmission of HIV and ZIKV 
between women co-infected with HIV and ZIKV, and women infected 
with either HIV or ZIKV alone.  
6. To compare the incidence of congenital malformations and other adverse 
outcomes (including microcephaly, neonatal death, central nervous system 
(CNS) malformations, hydrops and ocular abnormalities) among offspring 
of women co-infected with HIV and ZIKV, women infected with either 
HIV or ZIKV alone and doubly uninfected women.  
7. To compare the long-term effects (on growth, hearing, vision and 
neurodevelopment) of co-infection among children with in utero exposure 
to HIV and ZIKV, in utero exposure to either HIV or ZIKV alone and no 
in utero exposure to either virus. 

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Secondary 
objectives: 
1. To compare the severity of maternal and infant outcomes of ZIKV 
infection in symptomatic and asymptomatic women. 
2. To determine if co-infections and cofactors (including social and 
environmental factors) contribute to the presence of CNS malformations 
and if these factors influence the severity of adverse outcomes in the 
offspring.  
Evaluations: 
Maternal: 
Study visits will occur once per trimester during pregnancy, at delivery and 
approximately six weeks postpartum. At baseline, demographics will be 
collected and a targeted physical examination (PE) will be conducted. Baseline 
and interim medical, medication, substance use and other risk factor history will 
be obtained by interview and medical record abstraction. Medical history will 
include, but is not limited to, a recent history of symptoms consistent with ZIKV 
infection, including in their sexual partners and household members.  
At all visits, an assessment of symptoms suggestive of ZIKV will be targeted 
(i.e., fever, rash, arthalgias, myalgias, pruritus, headaches, eye pain and 
conjunctivitis), lymphadenopathy and a targeted PE performed. 
Fetal US will be performed (or data abstracted from clinically performed US) in 
the first, second and third trimesters.  
At each visit, HIV-infected women will have all available HIV viral loads 
(VLs), CD4+/CD8+ T-cells counts and percentages (T-cells) abstracted from 
their medical records. If these laboratory results are unavailable, blood samples 
will be collected for these laboratory assessments.  
Testing will be performed at each visit for the diagnosis of ZIKV infection. 
Tests will be performed to evaluate co-infections such as Dengue virus (DENV) 
and possibly other flaviviruses and other infections. Testing for environmental 
contaminants associated with morbidity and mortality in the developing fetus 
and infant at birth, such as lead, may be performed.  
Infant Participants: 
Data collected will include basic demographics and birth history. 
PEs will be conducted at birth and each study visit (or information abstracted 
from medical records if a PE was performed within the prior four weeks) and 
will consist of temperature, cardiac, respiratory, organ systems (hepatomegaly, 
splenomegaly), skin (petechial, rash, skin scarring), gastrointestinal (feeding, 
emesis, diarrhea) and neurologic examinations (seizures, apnea, reflexes/tone, 
asymmetry, eye opening, basic movement).  
Growth parameters including weight, length/height and head circumference will 
be recorded at birth and each study visit (or information abstracted from medical 
records if performed within the prior four weeks). 
Hearing assessments and ophthalmologic evaluations to detect chorioretinitis or 
abnormalities in conjunctiva, lens or retina will be performed at birth and each 
study visit. If initial exams are abnormal, referral for additional audiology and/or 
ophthalmology testing will be done and results recorded (if available) from any 
tests done for clinical reasons.  

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Results of any imaging assessments done for clinical care will be recorded. 
These include any neuroimaging studies (US, computerized tomography (CT), 
or magnetic resonance imaging (MRI)) to detect neurologic abnormalities 
including intracranial calcifications and non-neurologic imaging (including but 
not limited to chest X-rays, abdominal X-rays, abdominal CT scans or MRIs). 
All ZIKV-exposed infants will have a cranial US within the first three months.  
General motor development markers will be assessed. Neurodevelopmental 
(ND) status will be assessed using normal developmental milestones tests 
according to the standard of care.  
Testing will be performed at birth and subsequent visits for the diagnosis of 
ZIKV infection. Testing for other infections, including congenital infections that 
could lead to adverse infant outcomes may also be performed. 
Data analysis: 
Epidemiological and statistical data analyses will be conducted to address the 
primary and secondary objectives described above.  
 

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Maternal Study Schema 
 
 
Screening 
Entry  
Enrollment  
Brazil & P.R. sites:  
HIV+ pregnant women 
U.S. sites:  
HIV+ and HIV-, at-risk for ZKV, pregnant women 
2nd & 3rd Trimesters 
(24 & 34 wks. GA) 
(+/- 2 weeks) 
Delivery 
6 weeks 
postpartum  
(+/- 7 days) 
With or without acute ZIKV symptoms and  
< 18 wks. GA. 
Must be < 18 wks. GA at Enrollment if does not have laboratory-confirmed acute ZIKV-infection. Fetal US 
with fetal heart tones (only if not performed for Screening). Vital Signs (VS) & targeted PE. Zika-like 
symptoms, health, medication, pregnancy and other health risk factors questionnaires. Blood for 
toxoplasmosis, rubella, cytomegalovirus (CMV) and herpes (TORCH), Rapid Plasma Reagin (RPR) (tested 
locally if not done for clinical care). Blood for ZIKV (and possible DENV and other flaviviruses) serology. 
Blood / urine for reflex ZIKV RNA detection. Blood (plasma) and urine for future tests (optional). Provide 
Zika-symptom diary with instructions. Educate on Zika prevention. If HIV-positive: Abstract all HIV VLs and 
T-cells since the closest date prior to the LMP (perform if not done within the past 12 wks.). Perform HIV VL 
if symptomatic, regardless of testing history.  
Conduct or obtain results from fetal US performed since the last study visit. Zika-symptom diary review. 
Interim Zika-like symptoms, health, medication, pregnancy and other health risk factors questionnaires. VS & 
targeted PE. Re-educate on Zika prevention. Blood for ZIKV (and possible DENV and other flaviviruses) 
serology. Blood / urine for possible reflex ZIKV RNA detection. Blood (plasma) and urine for future studies 
(optional). If HIV-positive: Abstract all interim HIV VLs and T-cells (perform tests if not done within prior 
12wks.). Perform HIV VL if symptomatic, regardless of testing history. 3rd trimester only: Blood for TORCH, 
RPR (tested locally) (do not repeat previously positive tests).  
Obtain results from fetal US performed since the last study visit. Zika-symptom diary review. Interim Zika-like 
symptoms, health, medication, pregnancy and other health risk factors questionnaires. VS & targeted PE. Re-
educate on Zika prevention. Blood for ZIKV (and possible DENV and other flaviviruses) serology. Blood / 
urine for reflex ZIKV RNA detection. Blood (plasma) and urine for future studies (optional). Cord blood & 
placental tissue for future analysis (optional). (NOTE: Some cord blood will be used for real-time ZIKV serology 
if peripheral blood from infant is unobtainable.) If HIV-positive: Abstract all interim HIV VLs and T-cells 
(perform tests if not done within prior 12wks.). Perform HIV VL if symptomatic, regardless of testing history. 
Zika-symptom diary review. Interim Zika-like symptoms, health, medication and other health risk factors 
questionnaires. VS & targeted PE. Re-educate on Zika prevention. Blood for ZIKV (and possible DENV and 
other flaviviruses) serology. Blood/urine for possible reflex ZIKV RNA detection. Blood (plasma) and urine 
for future studies (optional). If HIV-positive: Abstract all interim HIV VLs and T-cells (perform tests if not 
done within prior 12wks.). Perform HIV VL if symptomatic, regardless of testing history. 
> 18 wks. GA, acute ZIKV symptoms and 
confirmed ZIKV-infection by ZIKV RNA 
detection. 
Written IC (or assent and parent(s)/legal guardian(s) permission, if warranted) for current maternal and future 
unborn infant’s enrollment (if possible). Verify the following: Age > 15 yrs. old and  
pregnant (fetal US with fetal heart tones or serum / urine βhCG). Obtain Race/Ethnicity. 

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Maternal Study Schema  
(ZIKV-Symptomatic or Asymptomatic with Positive Serology) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Women Experiencing Signs / Symptoms 
Consistent with Acute ZIKV-like Illness at 
Any Time While On Study  
Women with Positive ZIKV Serology at Any 
Study Visit, But without Clinical Signs / 
Symptoms of Acute ZIKV-like Illness 
NOTE: If ZIKV-like symptoms occur between 
scheduled study visits, have the participant come 
in for an unplanned, symptomatic visit assessment. 
 
Zika-symptom diary review. Interim health and 
medication history. 
Record signs and symptoms of ZIKV-like illness 
(i.e., fever, rash, arthalgia, myalgia, pruritus, 
headache, eye pain and conjunctivitis). Also, 
include presence of lymphadenopathy. 
Perform VS & targeted PE 
Pregnancy questionnaire 
Other risk factors questionnaires: If regularly 
scheduled study visit. If unplanned symptomatic 
visit, administer at continental U.S. sites only. 
If HIV-positive: Abstract all interim HIV VLs and 
T-cells. 
Collect biological samples: Blood for serum ZIKV 
& DENV (and other possible) serology; serum / 
urine for ZIKV RNA detection test. 
 If ZIKV serology positive or ZIKV RNA 
positive, perform (urine/serum) ZIKV RNA 
test from most recent prior visit.  
 If ZIKV serology negative, continue doing 
ZIKV serology at subsequent visits.  
 If ZIKV serology positive, continue doing 
ZIKV serology at subsequent visits until 
negative. 
 If ZIKV RNA positive, continue doing ZIKV 
RNA testing at subsequent visits until negative. 
 Blood for TORCH (if non-immune) and RPR 
to local laboratory, if clinically indicated.  
 Biorepository (if consented for future use): 
Blood (plasma) and urine.  
 Request other laboratory testing as part of 
standard of care. 
 If HIV-positive: HIV VL. 
Record results from interim fetal US performed 
according to country and local clinical guidelines 
since the prior study visit. 
Test available blood / urine samples as follows: 
 Serum/urine for ZIKV RNA detection on 
samples from the current visit and from the most 
recent prior visit, if available.  
 If ZIKV RNA negative, perform serum DENV 
serology from current visit if resides in or recent 
travel to DENV-endemic region.  
 Continue doing ZIKV serology at subsequent 
visits until negative. 
 If ZIKV RNA positive, continue doing ZIKV 
RNA testing at subsequent visits until negative. 

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Infant Study Schema 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
HIV-infected 
ZIKV-infected 
HIV-infected 
ZIKV-uninfected 
HIV-uninfected 
ZIKV-infected 
HIV-uninfected 
ZIKV-uninfected 
 Obtain written consent (or confirm consent) for infant participation and review study visit requirements 
with the parent(s)/legal guardian(s), per local requirements.  
 Demographics and birth data; temperature, head circumference, general PE (or abstract data from PE 
conducted as part of clinical care). 
 Neurological examination (seizures, apnea, reflexes/tone, asymmetry, eye opening and basic 
movement). 
 Hearing assessment (if abnormal, refer to audiology for a full audiological examination and obtain 
results from any tests done for clinical reasons). 
 Ophthalmologic evaluation (if abnormal, refer for additional testing and obtain results from any tests 
done for clinical reasons). 
 Obtain results from all neuroimaging assessments done for clinical reasons (head US, head CT/MRI 
scan). 
 Biological samples: Peripheral blood for ZIKV serology and possible DENV and other serology (as 
needed) and/or (urine/serum) ZIKV RNA detection, as indicated, urine for future testing (if consented 
for future use), TORCH and syphilis (RPR) (only if clinically indicated) and tested locally.  
 Remnant cerebrospinal fluid (CSF) sample for storage in biorepository, only if obtained for clinical care 
(if consented for future use). 
 Abstract HIV-related test results. 
Birth (within 48 hours) 
Months 3, 6, 12  
(12, 24 and 52 weeks (+/- 2 weeks)  
 Temperature, growth parameters & head circumference.  
 General PE (or abstract data from PE conducted as part of clinical care if performed within prior 4 
weeks). 
 Neurological examination (seizures, apnea, reflexes/tone, asymmetry, eye opening, and basic 
movement). 
 ND assessment for screening options (Hammersmith Infant Neurological Examination (HINE), Ages 
and Stages Questionnaire (ASQ), Bayley Scales of Infant and Toddler Development (BSID-III). 
 ND assessment for diagnostics (BSID-III) if screening is abnormal.  
 Hearing assessment (if abnormal, refer to audiology for a full audiological examination and record 
results). 
 Ophthalmologic evaluation (if abnormal, refer for additional testing and obtain results from any tests 
done for clinical reasons). 
 Obtain results from all neuroimaging assessments done for clinical reasons (head US, head CT/MRI 
scan). Zika-exposed infants only: Perform a head US for the study at the 3 month visit only if none 
was performed for clinical care by 3 months of age.  
 Biological samples: Blood and urine for ZIKV serology and possible DENV and other serology (as 
needed) and/or urine/serum ZIKV RNA detection, as indicated, urine for future testing (if consented 
for future use). 
 Remnant CSF sample for storage in biorepository, only if obtained for clinical care (if consented for 
future use). 
 Laboratory results abstraction from tests performed for clinical care. 
 HIV-exposed infants only: Abstract HIV-related test results and treatments, if applicable. 

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1.0 
Introduction 
1.1 
Background on ZIKV 
1.1.1 
Virology, History and Epidemiology of ZIKV 
ZIKV is a vector-borne single-stranded RNA virus of the genus Flaviviridae.[1] This 
virus family also includes other human pathogenic viruses such as DENV, West Nile 
Virus (WNV), Japanese encephalitis virus, Yellow Fever Virus (YFV), and tick-
borne encephalitic virus. ZIKV has both Asian and African lineages and is closely 
related to DENV. While ZIKV has been isolated from many species of Aedes 
mosquito, Aedes aegypti is thought to be the principal vector spreading ZIKV during 
the recent outbreaks in Latin America, the Caribbean and the continental U.S. 
ZIKV was first identified in the Zika forest near Kampala, Uganda in rhesus 
macaques in 1947.[2] Between the first isolation in monkeys until 2007, reports of 
human cases were rare and sporadic.[1] Although the virus had been isolated widely in 
Africa and Southeast Asia, the infection was largely asymptomatic, and no epidemics 
were observed. In 2007, an outbreak of the Asian lineage of ZIKV occurred in the 
Federated States of Micronesia.[3] Reports suggest that 73% of the population was 
infected within 3 years with 49 confirmed cases based on RNA detection or serologic 
criteria. In 2013, another outbreak involving 5,895 suspected cases of ZIKV and 294 
confirmed cases occurred within a 3-month period in French Polynesia.[4]  
In May 2015, the first cases of ZIKV were reported in northeast Brazil where the 
Asian lineage of ZIKV was confirmed.[1] Because of the lack of immunity in the 
Brazilian population and the abundance of Aedes aegypti mosquitos, an 
autochthonous transmission was established with ensuing rapid and large spread of 
ZIKV. Although initially concentrated in Brazil, the virus spread throughout South 
America, the Caribbean, and P.R. As of December 15, 2016, 48 countries in the 
Americas have reported vector-borne transmission of ZIKV.[5]  
Until recently, most infections from ZIKV were thought to be self-limiting and mild 
with resolution of most clinical findings by seven days in the small proportion of 
patients who were symptomatic.[6] However, during 2013-2014, the occurrence of 
Guillain-Barré syndrome (GBS) as a post-infectious complication of ZIKV infection 
was observed during the French Polynesian outbreak.[1] According to the most recent 
Pan American Health Organization (PAHO) report, 13 countries in the Americas 
including Brazil, Colombia, El Salvador and Venezuela have reported increases in the 
number of GBS cases with ZIKV confirmation.[5]  
With introduction of ZIKV, Brazil also witnessed an epidemic of microcephaly cases 
among newborns. According to the most recent PAHO report, 10,441 suspected cases 
of microcephaly and other CNS malformations were reported in Brazil during 2015-
2016, as compared to an annual average of 163 cases of microcephaly from 2001 to 
2014.[7] To date, the Brazilian Ministry of Health has confirmed microcephaly in 

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2,228 of these cases. Of the remainder, 5,040 were discarded because they had non-
infectious causes or did not meet the case definition and 3,173 are still under 
investigation. Microcephaly and other CNS malformations have also been noted in 
35% of reported miscarriages and stillbirths in Brazil during this period. To date, 22 
countries and territories in the Americas (including the U.S. and P.R.) have reported 
confirmed cases of birth defects and other abnormalities associated with ZIKV 
infection.[5] These numbers are expected to increase over the coming year as 
previously and newly exposed women complete their pregnancies.  
In the U.S., 4,809 cases of ZIKV with illness onset from January 1, 2015 through 
December 28, 2016 were reported to the national arbovirus surveillance system 
(ArboNET).[8] While these cases come from virtually the entire country, over half 
have come from only four states: New York (21%), Florida (18%), California (9%) 
and Texas (6%). The vast majority of cases (95.5%) have been associated with travel 
to an affected area; another 4.5% were acquired through local mosquito transmission, 
and < 0.1% have been associated with sexual contact with a traveler to an affected 
area. To date, local mosquito transmission in the U.S. has occurred in Miami-Dade 
County, Florida and in Brownsville, Texas.[9, 10] The extent of the ZIKV outbreak is 
far greater in the U.S. Territory P.R. where 33,865 locally acquired cases (through 
mosquito and sexual transmission) and 129 travel-associated cases were reported to 
ArboNET as of December 28, 2016.[8] Furthermore, 1,246 pregnant women in the 
U.S. and 2,701 pregnant women in U.S. territories (mainly P.R.) have been reported 
with laboratory evidence of possible ZIKV infection.[11]  
1.1.2 
Modes of ZIKV Transmission 
While ZIKV is spread primarily through bites of infected Aedes mosquitos, other 
forms of transmission have been described. In particular, several case reports have 
documented that ZIKV can be sexually transmitted from an infected male or female 
to his or her sexual partner, even if the infected individual is asymptomatic.[12-14] 
Supporting these data is evidence of ZIKV in the semen of infected males lasting as 
long as three months after symptom onset and the presence of infection in vaginal 
swabs.[15, 16] Other documented routes of ZIKV transmission include blood resulting 
in transmission through transfusions[17] and perinatal transmission from mother-to-
child.[18] While ZIKV has been detected in saliva, urine, breast milk and conjunctival 
fluid of infected humans,[19-21] there is limited data documenting transmission via 
these body fluids, though a recent report suggests transmission through contact with 
highly infectious body fluids in the hospital setting.[22]  
1.1.3 
U.S. Clinical Testing Guidelines for ZIKV  
In July 2016, the Centers for Disease Control and Prevention (CDC) issued guidelines 
for testing pregnant women who have had possible exposure to Zika and have 
symptoms consistent with ZIKV infection.[23] Possible exposure is defined as travel to 
or residence in an area with active ZIKV transmission, or sex with a partner who has 
traveled to or resides in an area with active ZIKV transmission without using 
condoms or other barrier methods to prevent infection. The recommended tests follow 

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a complicated algorithm (see 
http://www.cdc.gov/mmwr/volumes/65/wr/mm6529e1.htm) that includes real-time 
RT-PCR testing of serum and urine, a ZIKV Immunoglobulin M (IgM) antibody (Ab) 
test, and a plaque reduction neutralization (PRNT) test to rule out a false positive IgM 
result. However, a recent update to CDC laboratory testing guidelines does not 
recommend the use of PRNT for confirmation of ZIKV infection in P.R.[24]  
On the other hand, no testing is currently recommended for women who are 
contemplating pregnancy.[25] Instead, they are advised to consider avoiding non-
essential travel to areas with ZIKV transmission and, if they have traveled to or live 
in an area with ZIKV, to take steps to prevent mosquito bites and exposure through 
sex. The latter includes waiting at least eight weeks since last exposure before trying 
to conceive. The CDC also recommends that men who may have been exposed to 
ZIKV have protected sex for at least six months after the start of symptoms or last 
exposure.  
1.1.4 
Clinical Sequelae of ZIKV Infection During Pregnancy 
Microcephaly was not reported in association with ZIKV infection prior to the 
outbreak in the Americas. However, this association was not examined systematically 
during previous outbreaks. Recently, Besnard et al. reported a summary of findings 
from a retrospective review of the data from the 2013-2014 ZIKV outbreak in French 
Polynesia.[18] They describe 10 cases of congenital cerebral malformations in fetuses 
and newborns including 8 with major brain lesions and severe microcephaly, 6 with 
severe cerebral lesions without microcephaly and 5 with brainstem dysfunction 
without visible malformations. Among amniotic fluid samples that were collected 
between 20 and 29 weeks gestation from 7 cases, ZIKV RNA or infectious ZIKV 
isolates were detected in 4 of 5 microcephalic, but not in 2 normocephalic infants 
with severe brain lesions. 
In January 2016, one of the first reports of microcephaly cases in the current epidemic 
in Brazil was published.[26] All 35 infants met the case definition of a head 
circumference > 2 standard deviations below mean for sex and GA, and 74% of their 
mothers reported a rash illness during pregnancy. The CSF of affected infants was 
being tested for ZIKV at the time of publication. In another Brazilian study of 
ophthalmologic findings associated with ZIKV infection, 29 infants with 
microcephaly and presumed intrauterine ZIKV infection were examined. Ocular 
abnormalities were found in 10 children.[27] Over the past year, additional case series 
have been reported linking prenatal ZIKV infection to an increasing number of 
abnormalities in infants including joint contractures, clubfoot, and hearing loss.[28, 29] 
More recently, formal epidemiological studies have found further evidence of adverse 
birth and pregnancy outcomes associated with prenatal ZIKV infection. Preliminary 
findings from an ongoing case-control study in Brazil reported that mothers of 41% 
newborns with microcephaly vs. 0% of unaffected controls, had laboratory-confirmed 
ZIKV infection (crude odds ratio (OR): 55.5, 95% confidence interval (CI): 8.6 to 
infinity).[30] Another Brazilian study of symptomatic pregnant women found that 46% 

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of ZIKV-exposed pregnancies vs. 11.5% of unexposed pregnancies had adverse 
pregnancy and infant outcomes, including miscarriage, fetal death, growth 
retardation, microcephaly and other CNS abnormalities.[31] Lastly, a study from the 
U.S. Zika Pregnancy Registry found that 6% of women with laboratory evidence of 
possible recent ZIKV infection had offspring with ZIKV-associated birth defects, 
primarily microcephaly and brain abnormalities.[32] This proportion rose to 11% 
among women with symptoms or exposure exclusively during the first trimester. The 
prevalence of microcephaly among ZIKV-exposed women in this study is 
substantially higher than the background prevalence (7 per 10,000 live births) in the 
U.S.[33] 
1.2 
Background on HIV Infection in Pregnant Women 
Since the start of the HIV epidemic, there have been over 78 million people infected 
with HIV. Currently, more than 36 million people are living with HIV and 2 million 
new infections occurred in 2015.[34] In 2015, there were approximately 150,000 
children infected through mother-to-child transmission (MTCT), substantially fewer 
than in 2010 due to the fact that approximately 77% of HIV-infected pregnant women 
have access to ART during pregnancy, which has become standard of care 
worldwide.[34]  
The first cases of Acquired Immunodeficiency Virus (AIDS) in Brazil were reported 
in 1982, and Brazil was one of the first countries to provide universal access to 
ART.[35] In 2015, Brazil had a total of 830,000 people with HIV, and set a record for 
the number of people initiating ART (81,000), resulting in more than 455,000 people 
with HIV on ART. 
In the U.S., approximately 8,500 HIV-infected women give birth each year.[36] The 
standard of care in the U.S., as well as throughout the world, is to provide ART for 
both the health of the woman and to reduce the risk of MTCT. With the combination 
of routine HIV testing of pregnant women and access to ART, the MTCT rate has 
dropped to well below 1%. In the Pediatric HIV/AIDS Cohort Study (PHACS) - 
SMARTT, there have been 9 transmissions among more than 2,500 births in the past 
10 years.[37] While the use of ART during pregnancy has been a tremendous public 
health success, concerns still remain regarding both the short-term effects of HIV and 
ART during pregnancy as well as the longer-term effects among HIV-exposed 
uninfected (HEU) children.  
Given these concerns, the PHACS-SMARTT cohort study was initiated in 2007 to 
evaluate the effect of HIV infection and ART among HEU children. Many ART 
medications given to a pregnant woman cross the placenta and can be detected in the 
amniotic fluid and cord blood resulting in substantial fetal exposure.[38] Therefore, 
there is concern about toxicity of the drugs in the fetus and infant. It is noteworthy 
that none of the currently approved ART medications for the prevention of MTCT of 
HIV are in Food and Drug Administration (FDA) Pregnancy Category A (no fetal 
risk ascertained in adequately controlled human studies). Thus, there is continued 
need to examine the toxicity of ART in HIV transmission prevention for the short-

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term toxicity of newer agents as well as the unanswered questions of longer-term 
toxicity and subtle adverse effects.  
1.2.1 
Short-term Toxicity of ART to the Fetus and Infant 
The first major study of ART use to prevent maternal transmission of HIV (the 
Pediatric AIDS Clinical Trials Group (PACTG) 076 Study) documented little short-
term toxicity beyond transient anemia.[39] Since then, a number of increasingly 
complex regimens were studied for HIV treatment efficacy during pregnancy for the 
mother as well as for reducing transmission to the infant. These included studies that 
utilized nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse 
transcriptase inhibitors (NNRTIs) and protease inhibitors. The short-term toxicity 
risks for the fetus and infant were low; therefore, HIV transmission prevention by the 
use of ART has become standard of care and has been thought to have an acceptable 
risk profile in the short term. However, PHACS and others have demonstrated an 
increased risk of preterm birth with exposure to protease inhibitor-based combination 
antiretroviral therapy (cART) early in pregnancy.[40] 
1.2.2 
Long-term Toxicity of ART to the Infant 
A number of possible toxicities have been reported in ART-exposed children with 
some of the major areas being growth, neurologic, ND and cardiac. These and other 
toxicities need examination. The SMARTT study uses an innovative trigger-based 
surveillance approach for identifying AEs that may be attributed to prenatal and 
perinatal ART exposure. All children receive periodic clinical and laboratory 
evaluations and only those meeting certain thresholds or “triggers” receive additional 
assessments to evaluate whether or not they meet specific AE case criteria. We 
demonstrated that this approach estimates AE rates more efficiently than evaluating a 
random sample of the same size, and yields unbiased estimates of AE rates after 
correcting for the sensitivity of the trigger to identifying AEs.[41] To date, 49% of 
participants have met a trigger and 25% met case status (an AE) in ≥ 1 domains.[42] 
We identified several signals suggesting specific AEs from ART exposure that 
warrant further evaluation. For example, zidovudine (ZDV) was associated with 
higher risk of a metabolic case, characterized as high body mass index and abnormal 
lipid levels and/or insulin resistance. The combination of didanosine plus stavudine 
was also associated with higher risk of both neurologic and language cases. In other 
SMARTT-related research studies, we also found that atazanavir was associated with 
lower language achievement at one year of age and a two-fold higher risk of 
congenital anomalies.[43-45] 
1.2.3 
Neurologic, ND, Behavioral, and Language Dysfunction 
French studies[46] have suggested that perinatal exposure to ART nucleoside 
analogues may lead to persistent mitochondrial dysfunction. Mitochondrial disorders 
encompass a broad spectrum of clinical disease that include neurologic or ND 
abnormalities. The French studies specifically reported an increased prevalence of 
both severe and mild/moderate neurobehavioral abnormalities. Furthermore, they 

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reported an increased prevalence of febrile seizures associated with perinatal 
exposure to ARV nucleoside analogues in uninfected but exposed children.[47] In 
contrast, a study from the U.S. suggested that there is no global effect of ZDV on 
neurodevelopment up to four years of age.[48, 49] However, there are sporadic reports 
of mitochondrial-like disease with ragged red fiber abnormalities on muscle biopsy 
and developmental and visual disturbances,[50] as well as reports of CNS MRI 
abnormalities in children prenatally exposed to ART.[51] 
HIV and HIV-associated treatment exposures during gestation of uninfected infants 
may also affect neurobehavioral development and result in more subtle abnormalities 
that may not necessarily be related to mitochondrial function or birth defects.[47] The 
Women and Infants Transmission Study[52] showed that among HEU children up to 
24 months of age, mental and motor development scores were lower among older 
children, both with and without prenatal exposure to illicit drugs. The extent of this 
decline and decreased levels of functioning (more than 8% scoring > 2 standard 
deviations below expected) are significantly greater than were observed in a similar 
sample of unexposed children of low socioeconomic and minority status.[53] The 
etiology of this ND decline and impaired level of functioning has not been elucidated, 
but factors that may play a role are time of exposure to HIV, ART, other HIV-
associated medications, illicit drug use and/or other environmental toxin exposure.  
PHACS has shown that HEU youth are at increased risk for poor ND outcomes.[44, 54-
58] However, no specific ARTs or classes have been clearly implicated. PHACS found 
a high risk of late language emergence among HEUs at both one (26%) and two 
(23%) years of age, with risk at one year of age associated with atazanavir 
exposure.[43, 44] PHACS is currently exploring the hypothesis that atazanavir results in 
chronic fetal exposure to modest elevations of bilirubin.[59]  
Different aspects of the nervous system may be at risk of insult during the successive 
gestational periods because of the course of prenatal development.[47] A number of 
studies have shown that insults, such as maternal infection, particularly during the 
second trimester, are associated with neuropsychiatric abnormalities,[60] while insults 
during the first trimester seem more associated with abnormalities in global structure 
of the brain.[61] The effects of illicit drug abuse during pregnancy are well 
documented and may interact with the effects of HIV infection and ART providing 
additive negative effects on neurobehavioral development. 
1.2.4 
Hearing Dysfunction 
While the majority of cases of moderate to profound permanent hearing loss with 
congenital and neonatal onset are identified by direct screening,[62-64] some cases are 
missed by otoacoustic emissions (OAEs) and/or automated auditory brainstem 
response (AABR) hearing screening technologies in the newborn period. This is 
because the hearing impairment is mild in degree,[64, 65] unusual in configuration[66] or 
late in onset.[67] Infants who fail hearing screening or children who are later identified 
with risk indicators for hearing loss (e.g., speech and language delay or family 

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concern), require referral for comprehensive audiometric examination to determine 
hearing status and the need for follow-up treatment/testing.[68]  
A challenge in studying hearing in infants and children who were prenatally exposed 
to ART is that maternal HIV infection can be associated with conditions that affect 
hearing in the infant. Infants of mothers living in poverty are at increased risk of 
prematurity, very low birth weight and other maternally transmitted infections such as 
syphilis, toxoplasmosis, and CMV. These conditions are known to be associated with 
increased risk for hearing loss in the newborn period.[68-70] In contrast, other 
exposures that occur in HIV-infected mothers such as prenatal drug and alcohol 
exposure have not been associated with sensorineural hearing loss in the newborn.[71, 
72] 
Overall, PHACS found that 3.1% of infants in SMARTT had an abnormal newborn 
hearing screen, with a lower risk found with first trimester tenofovir exposure.[73] 
1.2.5 
Consequences of Prenatal HIV Infection on Immunity and Impacts of Co-Infections 
on Mother and Fetus  
To promote and support pregnancy and the growing fetus, an immunological 
adaptation during pregnancy is needed. This sanctuary is damaged when this 
protection is broken by a viral infection and so adverse outcomes may result such as 
increased maternal morbidity related to the infection, propagation of other 
microorganisms, fetal demise, preterm labor and MTCT of viral and other agents.[74] 
HIV infection is known to have deleterious effects on the immune system, mainly on 
the CD4+ T-cells compartment, B-lymphocytes and Ab responses to pathogens and 
vaccines.[75] These alterations are linked to immune activation/dysregulation and 
chronic inflammation.[76] When used correctly, ART results in rapid control of HIV 
with VL suppression in blood and partial restoration of immune function, thereby 
preventing the various complications that define AIDS. However, even after 
treatment, HIV infection evolves into a chronic condition with the potential to 
continuously affect the host immune system.[77] These facts are especially important 
to the pregnant mother and fetus when the pregnancy is threatened by a superimposed 
latent or acutely acquired infection.  
Increased risk of severe illness during pregnancy from viral infections has been 
reported during pandemics of influenza, Ebola, and Lassa fever, even in HIV-
uninfected women.[74, 78] Regarding ZIKV infection in pregnancy, apparently, no 
enhanced risk of morbidity or mortality in HIV-uninfected women has been described 
so far.[79] Nevertheless, only two cases of ZIKV infection in HIV-infected individuals 
have been reported to date: an adult male who had only mild symptoms and recovered 
well,[80] and an immunosuppressed pregnant woman who was using a combination 
ARV regimen and presented with a mild disease. While the pregnant case had a rapid 
and complete recovery, her severely affected fetus died at 20 weeks gestation.[81] 
Considering the potential of more severe disease in HIV-infected mothers, it is very 

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relevant to examine pregnant women with both ZIKV and HIV infections to assess 
the consequences of co-infection on the mother’s health.  
Moreover, it is possible that ZIKV may act as an HIV facilitator pathogen. That is, it 
could enhance HIV replication during infection due to the release of cytokines that 
can activate CD4 cells offering the inflammatory microenvironment, or it could 
directly bind to HIV proteins that support HIV replication.[82] High maternal HIV 
load is the strongest risk factor for MTCT of HIV, and reduction of HIV load with 
ARV significantly reduces risk.[83] Possibly, any infection that increases HIV VL in 
plasma, the genital tract or breast milk may indirectly raise the risk of MTCT of HIV. 
In addition, HIV’s systemic immunological activation consequences and impairment 
of the placental barrier could boost the risk of MTCT of the infecting agent, including 
ZIKV.  
The potential facilitation of MTCT of HIV and another virus in the presence of co-
infections with several microorganisms during gestation, before and after the era of 
ARVs, has been investigated in many studies. It has been shown that placental 
malaria is associated with increased MTCT of HIV, even at low maternal HIV 
loads.[84] Syphilis, a common co-infection in HIV-infected women, can also facilitate 
in utero transmission of HIV to infants.[85] Similarly, type 2 Herpes Simplex Virus co-
infection has been associated with increased intrapartum HIV transmission.[86] 
Although the Hepatitis B Virus (HBV) infection does not seem to be independently 
associated with increased transmission of HIV, maternal HIV/HBV co-infection may 
increase HBV transmission to the infant.[83] In addition, HIV/Hepatitis C Virus 
(HCV) co-infected mothers are at increased risk for perinatal transmission of both 
viruses.[87, 88] In the same direction, the prevalence of congenital CMV infection 
among infants born to HIV-infected women is higher (2% to 7%) as compared to the 
general newborn population (0.7%).[89] Although there are data suggesting that highly 
active antiretroviral therapy (HAART) decreases the risk of congenital CMV 
infection among HIV-infected mothers,[90] a more recent report shows that congenital 
CMV infection remains higher among infants born to HIV-infected mothers receiving 
ARVs.[91] 
In contrast to HIV, the correlates of MTCT of ZIKV and the fetal disease caused by 
ZIKV infection are currently unknown. However, it is likely that the immune system 
dysfunction of HIV-infected pregnant women potentially enhanced by acute viral 
infection, even in the presence of ARVs, could increase the risk of MTCT of both 
HIV and ZIKV and their consequences.  
1.3 
Rationale for HIV ZIP Study 
Remarkable strides have been made in the last decade in the treatment of HIV-
infected pregnant women, suppressing HIV RNA levels, which result in immune 
restoration for their own health and the prevention of MTCT of HIV. The occurrence 
of ZIKV infection among HIV-infected pregnant women raises serious concerns 
regarding the ARVs ability to suppress HIV RNA levels and the risk of MTCT of 
both viruses. There is also a growing number of significant adverse infant outcomes 

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following prenatal ZIKV infection, and given the fact that HIV is also a neurotropic 
virus, the ability of an HIV-infected pregnant woman to maintain HIV RNA 
suppression will be critical in regards to her child’s health. Therefore, there is an 
urgent need to formally investigate both maternal and child effects of HIV and ZIKV 
co-infection in pregnant women. Delineation of adverse effects would allow 
formulation of standard-of-care recommendations to minimize adverse effects but 
enable continuation of preventive therapy. 
2.0 
Study Objectives 
2.1 
Primary Objectives  
2.1.1 
To determine the feasibility of enrolling pregnant women with ZIKV/HIV co-
infection, HIV infection alone, ZIKV infection alone as well as doubly uninfected 
women into a prospective cohort study at selected sites in the continental U.S., P.R. 
and Brazil.  
2.1.2 
To compare HIV viral suppression in HIV-infected women with and without ZIKV 
co-infection during pregnancy and the time of delivery. 
2.1.3 
To compare the incidence of ZIKV infection among pregnant women with HIV 
infection and those without HIV infection.  
2.1.4 
To compare the incidence of adverse pregnancy outcomes between women co-
infected with HIV and ZIKV, women infected with either HIV or ZIKV alone and 
doubly uninfected women.  
2.1.5 
To compare the incidence of vertical transmission of HIV and ZIKV between women 
co-infected with HIV and ZIKV, women infected with either HIV or ZIKV alone.  
2.1.6 
To compare the incidence of congenital malformations and other adverse outcomes 
(including microcephaly, neonatal death, CNS malformations, hydrops and ocular 
abnormalities) among offspring of women co-infected with HIV and ZIKV, women 
infected with either HIV or ZIKV alone and doubly uninfected women.  
2.1.7 
To compare the long-term effects (on growth, hearing, vision and neurodevelopment) 
of co-infection among children with in utero exposure to HIV and ZIKV, in utero 
exposure to either HIV or ZIKV alone and no in utero exposure to either virus. 
2.2 
Secondary Objectives  
2.2.1 
To compare the severity of maternal and infant outcomes of ZIKV infection in 
symptomatic and asymptomatic women. 
2.2.2 
To determine if co-infections and cofactors (including social and environmental 
factors) contribute to the presence of CNS malformations and if these factors 
influence the severity of adverse outcomes in the offspring.  

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3.0 
Study Design 
This is a two-phase prospective international cohort study of pregnant women/infant 
pairs whose goals are to compare the incidence of ZIKV infection among pregnant 
women with and without HIV infection and to determine the risk of adverse maternal 
and child outcomes associated with ZIKV/HIV co-infection across clinical sites in the 
continental U.S., P.R. and Brazil.  
Phase I will enroll pregnant women/infant pairs who are: (1) infected with HIV only; 
(2) infected with ZIKV only; (3) infected with HIV and ZIKV; and (4) not infected 
with either HIV or ZIKV, and will assess the feasibility of enrolling a total of 200 
pregnant women/infant pairs within a year, with a target of 150 HIV-infected women 
across all sites, 50 HIV-uninfected women from continental U.S. sites only and a 
minimum of 20 who are co-infected with HIV and ZIKV.  
Should the feasibility of Phase I prove successful, Phase II will commence by 
enrolling up to 1,800 additional pregnant women/infant pairs to the 4 groups 
described above. The comparison group of HIV-uninfected women/infant pairs from 
P.R and Brazil (ZIKV-infected and uninfected) will be obtained from data collected 
in the concurrent ZIP study.  
All HIV-infected and uninfected participants will be tested for ZIKV. Enrolled 
women will be followed throughout pregnancy and up to six weeks postpartum. 
Infants born to enrolled women will be followed for a full year after birth. Thereafter, 
the infants born to women at continental U.S. and P.R. sites also implementing the 
SMARTT protocol may be followed up yearly until adulthood through SMARTT, 
depending on availability of funds.  
3.1 
Study Population 
The study will enroll pregnant women/infant pairs, ages 15 years and older, who 
reside in a geographic area accessible to one of the selected clinical research sites in 
Brazil, P.R. and the continental U.S., meet the criteria for ZIKV infection risk, are 
less than 18 weeks GA and are HIV-infected or uninfected (the latter in the 
continental U.S. only).  
In addition to the above, the study will enroll HIV-infected and uninfected women 
(the latter in the continental U.S. only) at 18 weeks or greater GA who present with 
acute ZIKV-like symptoms and have confirmed ZIKV infection by a positive ZIKV 
RNA detection test at the Screening visit.  
Lastly, all infants born to women while enrolled in this study will also be enrolled if 
the parent(s)/legal guardian(s) consents for the infant to participate. Infants may fall 
into one of the following groups: (1) HIV-infected only; (2) ZIKV-infected only; (3) 
HIV- and ZIKV-infected; and (4) HIV- and ZIKV-uninfected.  

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3.2 
Sample Size 
Phase I of the study will enroll 200 pregnant women at risk for ZIKV infection and 
their children born from that pregnancy to determine the feasibility of enrolling a 
target of 150 HIV-infected and 50 HIV-uninfected women within a year, and a 
minimum of 20 women who are co-infected with HIV and ZIKV. Should the 
feasibility of Phase I prove successful, Phase II will commence by enrolling up to 
1,800 additional pregnant women at risk for ZIKV infection and their children born 
from that pregnancy.  
4.0 
Protocol Registration Procedures 
Prior to implementation of this protocol, and any subsequent full version 
amendments, each site must have the protocol and the protocol consent form 
approved by its local IRB/IEC and any other applicable regulatory entity. The site 
must then register the protocol with the Westat Regulatory Affairs by completing the 
protocol registration checklist and submitting all required regulatory documents. 
Westat Regulatory Affairs will review the materials to ensure that the site has the 
necessary resources to fulfill study requirements and to follow federal and local 
IRB/IEC regulations. Sites may not begin enrollment prior to receiving registration 
approval from Westat Regulatory Affairs. All original approved documents must be 
maintained at the clinical site.  
The procedures for registration are outlined in the HIV ZIP MOP, which can be found 
in the HIV ZIP link on the NICHD Clinical Studies website 
(www.nichdclinicalstudies.org).  
5.0 
Selection and Enrollment of Study Participants  
5.1 
Maternal Inclusion Criteria  
Pregnant women who reside in a geographic area accessible to one of the selected 
clinical research sites are eligible for participation if the following criteria are met:  
5.1.1 
Provides written IC (or assent and parent(s)/legal guardian(s) permission, where 
required per state or country regulations). 
5.1.2 
Age 15 years or older at enrollment. 
5.1.3 
Confirmation of pregnancy by βhCG measurement in blood or urine or fetal US with 
fetal heart tones present. 
5.1.4 
Based on pregnancy calculator or fetal US: Confirmation of being at < 18 weeks GA 
of pregnancy or at any GA if presents with acute ZIKV-like symptoms (i.e., fever, 

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rash, arthralgia, myalgia, pruritus, headache, eye pain and conjunctivitis) and has 
laboratory-confirmed ZIKV infection by ZIKV RNA detection. 
5.1.5 
Plans on remaining in the area of the current study site or if moving, within an area of 
any other study site, for the duration of her and her child’s participation. 
5.1.6 
Willingness of parent(s)/legal guardian(s) to provide written consent to enroll the 
infant from the current pregnancy once delivered.  
In addition, at least one of the following three ZIKV-exposure risk categories must be 
met: 
5.1.6.1 
Has resided in for at least three months or traveled within the last three months to a 
country or U.S. Territory with active ZIKV transmission. The most up-to-date list of 
these locations can be found at http://www.cdc.gov/zika/geo/active-countries.html.  
(Within the U.S., the list of communities with local active ZIKV transmission can be 
found at http://www.cdc.gov/zika/intheus/maps-zika-us.html.) 
5.1.6.2 
Sexual partner has resided in or traveled within the last six months to a country or 
U.S. Territory with active ZIKV transmission, or was diagnosed with ZIKV within 
the previous six months; or 
5.1.6.3 
Household member has been diagnosed with ZIKV infection or has traveled since the 
woman’s LMP to a country or U.S. Territory with active ZIKV transmission.  
5.1.7 
HIV-infected women only: Laboratory evidence or clinical criteria for a confirmed 
case of HIV infection per CDC Surveillance Case Definition for HIV Infection, 2014 
(Section 1.11 or Section 1.1.2) 
http://www.cdc.gov/mmwr/preview/mmwrhtml/rr6303a1.htm. 
5.2 
Maternal Exclusion Criteria 
5.2.1 
Incarcerated or placed in detention. 
5.2.2 
Enrolled in other clinical research (including other ZIKV research) requiring blood 
collection, which in combination with HIV ZIP evaluations would exceed a total 
blood draw volume of 50 ml every 8 weeks and/or blood collection would be required 
more frequently than 2 times per week. 
5.3 
Infant Inclusion Criteria 
5.3.1 
Born to an enrolled mother. 
5.3.2 
Parent(s)/legal guardian(s) provided written IC for his or her child to participate. 
(Written IC for participation of the infant(s) born from this pregnancy may be 
obtained at the same time maternal consent is obtained or any time during the 
pregnancy and confirmed at delivery per local requirements.) 

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5.4 
Infant Exclusion Criteria 
5.4.1 
Enrolled in other clinical research (including other ZIKV research) requiring blood 
collection, which in combination with HIV ZIP evaluations, would exceed 3 ml per 
kg in an 8-week period and/or blood collection would be required more frequently 
than 2 times per week. 
5.5 
Participant Recruitment  
Participants will be recruited at implementing sites on an ongoing basis, whether from 
within their existing patient population or outside referrals. However, the protocol 
team will monitor enrollment of the HIV-negative maternal cohort, so as not to 
exceed the maximum goal of 50 participants during Phase I. A research staff member 
will contact potential participants (e.g., during a routine prenatal visit) and provide an 
overview of the study, including objectives, design and procedures.  
Each potential participant who has her medical records reviewed to assess potential 
eligibility, and is approached for recruitment and/or consented for study participation 
will be assigned a Screening Identification (SID) number. Any links between 
individuals’ names and their SID numbers will be maintained in a locked cabinet 
accessible to study staff only. When study accrual ends, site staff will obliterate all 
linked SID numbers belonging to individuals who did not consent to participate in the 
study.  
The following information will be recorded on an HIV ZIP Screening Form, which 
will be maintained in a secure area at the clinical research site and accessible only to 
study staff: Age, race, ethnicity, pregnancy status, LMP, HIV status, ZIKV-exposure 
risk and other non-personally identifying information (PII), and inclusion criteria that 
do not require study procedures to be performed. Individuals assessed as ineligible for 
enrollment will have the reasons for ineligibility recorded. Individuals who are 
approached, but do not consent to participate, will be asked if they are willing to 
supply their reason(s) for declining participation; responses will be recorded. 
Information collected on this form will be entered in the study database using the SID 
number. Names, dates of birth and other individually identifying information will not 
be recorded or entered into the study database.  
The protocol team may request tabulated information on individuals who participated 
in the recruitment process, but did not provide IC and the reasons not consented, as 
well as those that provided IC, but were deemed ineligible. These data will provide 
general information on the population that is recruited at the participating sites into 
the study.  
5.6 
Informed Consent  
Once it is determined that an individual may qualify for the protocol, study details 
will be discussed and all questions answered during the IC process. Written IC from 
the individual (or assent with parent(s)/legal guardian(s) permission, if applicable) 

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will be obtained before any study-related procedures or activities are performed (see 
attached separate document Appendix IV). Consent must be obtained prior to 
performing any study screening procedures.  
IC will consist of a description of the study’s purpose, study visits and procedures and 
evaluations to be performed, information that will be collected from their and their 
child’s medical records, possible risks and benefits and the voluntary nature of 
participating. Individuals who express interest in the study will be required to provide 
written IC (or assent with parent(s)/legal guardian(s) permission, if applicable) before 
any study-related procedures are performed. Eligibility criteria will be confirmed by 
study staff before enrolling into the study. A separate written IC will be obtained for 
the women and infant participants.  
5.7 
Co-enrollment Guidelines 
HIV ZIP sites that are also implementing the PHACS SMARTT study are encouraged 
to discuss co-enrollment of the women and their newborn infants into SMARTT. 
Participants that also consent to enroll into the SMARTT study will be advised 
through the IC process that data between the two studies will be shared. For women 
co-enrolled in HIV ZIP and SMARTT, sites should query the SMARTT Protocol 
Team at enrollment for guidance on modifying the SMARTT blood draws so as not to 
exceed the maximum total allowed.  
Enrollment into other studies is first at the discretion of the local Principal 
Investigator (PI). However, he or she must take into account any issues (such as blood 
volume and study burden) that enrollment in the additional study may cause, and 
which may compromise the site’s ability to fulfill the requirements of HIV ZIP. The 
approval of the HIV ZIP Protocol Co-Chairs and the Protocol Team must be obtained 
prior to co-enrollment into other studies. 
6.0 
Study Evaluations 
See Appendices I and II for the schedule of evaluations described below. Detailed   
guidelines for specimen collection, processing, storage and shipping can be found in 
the HIV ZIP MOP. 
6.1 
Maternal Evaluations 
6.1.1 
Baseline and Interim Medical and Medication History  
Information will be obtained by chart review and interviewing participants. 
Participants will be queried regarding a history of significant medical disorders of the 
head, eyes, ears, nose, throat, mouth, cardiovascular system, lungs, gastrointestinal 
tract, liver, pancreas, kidney, nervous system, blood, lymph glands, endocrine system, 
musculoskeletal system, skin and genital/reproductive tract. A history of any 
allergies, recent travel (of sex partner and household members), animal exposure, and 

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previous DENV, YFV or Chikungunya (CKNV) infections will be solicited. A recent 
history of symptoms consistent with ZIKV infections will also be solicited. A history 
of any prior infections with parvovirus, herpes and syphilis, among others, will be 
obtained. A vaccination history, as well as prescription and non-prescription 
medications, vitamins and herbal supplements taken since the woman’s LMP and 
throughout her pregnancy will be documented.  
6.1.2 
Targeted Physical Examination  
This examination will be conducted at each study visit beginning with the Entry visit, 
and will assess the woman’s general appearance and include an assessment of signs 
and symptoms of acute ZIKV infection (fever, rash, arthalgia, myalgia, pruritus, 
headache, eye pain, conjunctivitis), as well as lymphadenopathy. 
6.1.3 
Pregnancy Questionnaire  
Signs or symptoms of pregnancy complications will be elicited including decrease in 
fetal movement, rupture of membranes, vaginal bleeding, headaches, scotomata, 
nausea and vomiting.  
6.1.4 
Other Health Risk Factors  
Questions on cigarette, alcohol and illicit drug use, as well as environmental risk 
factors, including, but not limited to, residence and occupation, will be administered.  
6.1.5 
Imaging Assessments  
Fetal US will be performed (or data abstracted from clinically performed US) while 
the woman is pregnant. If US is performed more frequently for clinical management 
of the pregnancy, data from the US will also be recorded. 
6.1.6 
Laboratory Assessments  
 Confirmation of pregnancy by βhCG measurement in blood or urine will be done 
if no prior testing was performed or pregnancy not was not already confirmed by 
fetal US.  
 Assays to detect Abs or antigens (Ags) to TORCH will be run per local clinical 
laboratory procedures. The Rapid Plasma Reagin (RPR) tests will be performed 
on plasma in accordance with local clinical laboratory procedures. Because false 
positives may occur, additional testing may be required and conducted as part of 
the clinical care for the participant.  
 Testing will be performed for the diagnosis of ZIKV in samples from urine and 
blood. The specific laboratories that will be conducting the ZIKV assays as well 
as instructions for processing, handling, shipping, testing methods and storage of 
samples are maintained in the HIV ZIP MOP. 

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 Assays for the detection of Ab (Enzyme-linked immunosorbent assay (ELISA)) 
may be performed for DENV and in some cases for CKNV or WNV.  
 HIV-infected women will have all HIV VLs and T-cells performed since just 
prior to their LMP and throughout enrollment abstracted from medical records. 
HIV VL and T-cells will be done if no results from within the 12 weeks prior to a 
study visit are available. HIV VL testing will be performed if the woman has 
ZIKV-like symptoms, regardless of the availability of prior results in her medical 
record. 
NOTE: See the HIV ZIP MOP for the study assay details and algorithms, as well as 
the Laboratory Processing Chart (LPC) for specific laboratory methods for each assay 
and instructions for handling and storage of samples.  
6.1.6.1 
Detection of Anti-ZIKV Antibodies 
 Starting from the Entry visit, blood will be collected at each visit, to detect serum 
anti-ZIKV IgM Abs using standardized protocol and reagents for ZIKV. Anti-
ZIKV IgM Abs will be detected initially using laboratory tests such as the CDC 
qualified assay (ZIKV IgM Ab capture ELISA (MAC-ELISA)). As more 
specific/sensitive assays are developed, those assays may be substituted (the HIV 
ZIP MOP will be updated).  
6.1.6.2 
Detection of ZIKV RNA 
 If serum tests positive for anti-ZIKV IgM Abs at any study visit, RT-PCR (or 
other possible later developed ZIKV RNA detection test) to test for ZIKV RNA 
will be performed on serum and urine from that same collection date, as well as 
from the most recent prior study visit, if available. If serum tests positive for anti-
ZIKV IgM Abs and ZIKV RNA detection test is negative and the woman resides 
in or she/her sex partner has a recent travel exposure history to a DENV-endemic 
area, testing for anti-DENV IgM Abs (and possibly other flaviviruses) will be 
performed (serum) on the current visit’s samples, if available.  
 Specimens collected from women presenting with ZIKV-like symptoms at any 
visit will be processed immediately and have anti-ZIKV IgM Abs (serum), anti-
DENV IgM Abs (serum) and RNA detection for ZIKV (serum and urine) 
performed at the same time. If anti-ZIKV IgM Abs or ZIKV RNA test positive,  
ZIKV RNA detection testing will be performed (serum and urine) on the most 
recent prior visit (if available).  
 All sites will use a standardized protocol for RT-PCR testing (or other possible 
later developed ZIKV RNA detection test). Any remaining blood (serum) and 
urine samples will be temporarily stored for possible future testing pending 
serology results. 

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6.1.6.3 
Biorepository Specimens (if consented for future use) 
 At each study visit, plasma will be prepared from collected blood and additional 
urine will also be collected; both will be stored in the NICHD Biorepository to be 
used in future assays for assessment of the immune response to ZIKV as assays 
are developed. (Allowable future use will be documented in the consent for each 
site.) 
 At the time of delivery, cord blood and placental tissue will be collected for 
storage at the NICHD Biorepository for possible future ZIKV testing, when 
convenient.  
6.1.6.4 
Specimen Collection, Preparation, Handling and Shipping 
 Blood and urine samples that are not being tested locally will be received, 
processed, aliquoted and shipped for testing and temporary storage to the site’s 
assigned central laboratory according to standard laboratory procedures outlined 
in the HIV ZIP MOP.  
 Cord blood samples, as well as placental tissue, collected at the time of delivery 
for possible future testing will be received, processed, aliquoted (if applicable) 
and shipped to the NICHD Biorepository for temporary storage as instructed in 
the HIV ZIP MOP. If newborn peripheral blood is unobtainable, cord blood will 
be prepared and shipped to the site’s assigned central laboratory for real-time 
ZIKV testing, with remnant cord blood shipped for storage to the NICHD 
Biorepository. 
6.2 
Infant Evaluations 
6.2.1 
Baseline Demographics and Birth Information  
Data collected will include basic demographics and birth history (e.g., date of birth, 
sex, race, ethnicity) and delivery characteristics (e.g., GA, type of delivery, 
complications at delivery). 
6.2.2 
Physical Examination  
The examination will consist of temperature, cardiac, respiratory, organ systems 
(hepatomegaly, splenomegaly), skin (petechial, rash, skin scarring), gastrointestinal 
(feeding, emesis, diarrhea) and neurologic examinations (seizures, apnea, reflexes/tone, 
asymmetry, eye opening, basic movements). An examination does not need to be 
performed if required data from an examination performed within four weeks prior to 
the study visit can be abstracted from medical records.  
6.2.3  
Growth Parameters  
Growth parameters (weight, length/height and head circumference) will be recorded 
at birth and at each study visit. Growth parameters are not required at study visits if 

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they were obtained within four weeks prior to the study visit and the required data can 
be abstracted from medical records. A head circumference must be included in the 
examination. 
6.2.4  
Hearing and Ophthalmologic Assessments  
Hearing assessments (OAE) or AABR, general eye exams and ophthalmologic exams 
to detect chorioretinitis or abnormalities in the conjunctiva, lens or retina will be 
conducted. If initial exams are abnormal, referrals for additional audiology and/or 
ophthalmology testing will be done and results collected (if available) from any tests 
done for clinical reasons.  
6.2.5  
Imaging Assessments  
Results of any imaging assessments performed for clinical care will be recorded. 
These include any neuroimaging studies (US, CT or MRI) obtained for clinical 
reasons to detect neurologic abnormalities including intracranial calcifications. A 
head US will be performed at the 3 Month visit on ZIKV-exposed infants only if none 
was performed by the age of 3 months. In addition, data from non-neurologic imaging 
will also be recorded (including, but not limited to, chest X-rays, abdominal X-rays, 
abdominal CT scans or MRIs).  
6.2.6  
Neurodevelopmental Assessment  
Assessment of general motor development markers will be performed. 
Neurodevelopmental (ND) status of study participants will be assessed using normal 
developmental milestones tests as per standard of care. ND may also be assessed by 
administering the ASQ and the BSID, if clinically indicated and trained personnel are 
available. 
6.2.7  
Laboratory Assessments  
 Individual assays to detect Abs/Ags to TORCH and an RPR for syphilis at birth, 
if indicated, due to fetal and/or neonatal findings or maternal history (see the HIV 
ZIP MOP for a table on general guidelines for TORCH and syphilis testing for 
asymptomatic newborn infants according to maternal history) will be performed 
in accordance with local clinical laboratory procedures. Because false positives 
may occur, additional testing may be required and conducted as part of the 
clinical care for the participant.  
 Urine and peripheral blood will be obtained to assess for ZIKV at each study visit. 
(At the delivery visit, cord blood may be used immediately to assess for ZIKV if 
newborn peripheral blood is unobtainable, with remnant cord blood shipped for 
storage to the NICHD Biorepository (see Section 6.1.7.4)). The specific 
laboratories that will be conducting the ZIKV assays as well as instructions for 
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maintained in the HIV ZIP MOP, as well as the LPC for specific laboratory 
methods for each assay and instructions for handling and storage of samples. 
 Assays for the detection of Ab (ELISA may be performed for DENV, and in some 
cases, for CKNV and WNV) will be performed.  
 HIV-related testing results will be abstracted from medical records. 
6.2.8 
Biorepository Specimens (if consented for future use) 
 Urine for possible future testing will be stored. 
 Remnant CSF, if collected at any time for clinical care, will be stored. 
7.0 
Study Schedule and Procedures 
7.1 
Maternal Study Visits 
See Appendix I for the schedule of evaluations. Detailed guidelines for specimen 
collection, processing, storage and shipping can be found in the HIV ZIP MOP. 
Medical and medication history will be collected via medical record abstraction and 
participant self-report.  
7.1.1 
Screening Visit 
Study staff will review eligibility criteria and inform individuals about study 
objectives, design and procedures. Limited, non-PII will be recorded on the HIV ZIP 
Screening Form as described in Section 5.5. If deemed potentially eligible, 
individuals will be interviewed to evaluate their willingness to participate in the 
study. If interested, study staff will obtain written IC (or assent with parent(s)/legal 
guardian(s) permission) and will assign a unique Participant Identification (PID) 
number prior to performing any study-related procedures or evaluations. After written 
consent or assent with parent(s)/legal guardian(s) permission is obtained, the 
following will be completed: 
 Pregnancy confirmation by serum or urine βhCG or fetal US with fetal heart 
tones, if not already performed for clinical care or unable to access the results. 
 Date of birth. 
 If acute ZIKV-like symptoms are present and GA is > 18 weeks, collect the 
following biological samples: 
– Blood for ZIKV RNA detection and ZIKV serology.  
– Urine for ZIKV RNA detection. 

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– Blood for plasma for future studies (if consented for future use). 
– Urine for possible later testing (if consented for future use). 
7.1.2  
Entry Visit  
For the purposes of this study, enrollment will occur on the same day as, but prior to 
conducting the Entry visit. Verify eligibility criteria prior to study enrollment.  
 Women who present with acute ZIKV-like symptoms at the Screening visit and 
are 18 weeks or greater GA must have a positive ZIKV RNA test to be enrolled, if 
otherwise eligible.  
 Women who present with acute ZIKV-like symptoms at the Screening visit and 
are less than 18 weeks GA will be enrolled and have their Entry visit conducted 
on the same day as the Screening visit, if otherwise eligible.  
 Women less than 18 weeks GA who do not present with acute ZIKV-like 
symptoms at the Screening visit, ideally should be enrolled and have their Entry 
visit conducted on the same day as the Screening visit, but they may complete the 
Entry visit evaluations at any time prior to 18 weeks GA.  
 The following will be completed at the Entry visit:  
– Fetal US with fetal heart tones only if not already done for the Screening visit 
or clinical care.  
– Medical and medication history. 
– Pregnancy questionnaire. 
– Other risk factors questionnaire.  
– Obtain results from clinical laboratory tests conducted for clinical care.  
– HIV-infected participants: Abstraction of all HIV VLs and T-cells starting 
from the closest, but prior to, the woman’s LMP.  
– Height, weight and VS (including blood pressure and temperature). 
– Targeted PE.  
 The following biological samples will be collected:  
– Blood for TORCH testing and syphilis (RPR) for clinical care and send to the 
local laboratory. (Do not repeat any tests already performed for routine 
prenatal care.) 

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– Blood for ZIKV RNA detection. (Do not collect if already collected at the 
Screening visit (i.e., > 18 weeks GA and acute ZIKV-like symptoms)). 
– Blood for ZIKV serology. 
– Blood for DENV serology and other possible serology as needed (CKNV or 
WNV) if resides in or recent travel history to DENV-endemic region. (Do not 
collect if already collected at the Screening visit (i.e., > 18 weeks GA and 
acute ZIKV-like symptoms)). 
– Blood for plasma for future studies (if consented for future use). (Do not 
collect if already collected at the Screening visit (i.e., > 18 weeks GA and 
acute ZIKV-like symptoms)). 
– HIV-infected women only: Blood for HIV VL and T-cells only if not 
collected within the prior 12 weeks. (HIV VL testing is required if ZIKV-like 
symptoms are present, regardless of testing history.) 
– Urine for ZIKV RNA detection. (Do not collect if already collected at the 
Screening visit (i.e., > 18 weeks GA and acute ZIKV-like symptoms)). 
– Urine for possible later testing (if consented for future use). (Do not collect if 
already collected at the Screening visit (i.e., > 18 weeks GA and acute ZIKV-
like symptoms)). 
 Educate about ZIKV prevention and the signs and symptoms of acute ZIKV 
infection and instruct to notify the clinic immediately if experiencing these 
symptoms (or if sex partner or household member is experiencing these 
symptoms).  
 Provide diary and instruct on how to record ZIKV-like symptoms between visits. 
7.1.3 
Second and Third Trimester Visits  
 The following will be completed at the Second and Third Trimester visits, which 
should be conducted at 24 and 34 weeks GA (+/- 2 weeks), respectively:  
– Conduct or obtain results from fetal US performed since prior study visit. 
– Zika-symptom diary review.  
– Interim medical and medication history. 
– Interim pregnancy questionnaire. 
– Other risk factors questionnaire.  
– Obtain results from clinical laboratory tests conducted for clinical care.  

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– HIV-infected participants: Abstraction of all interim HIV VLs and T-cells 
results.  
– Weight and VS (including blood pressure and temperature). 
– Targeted PE.  
 The following biological samples will be collected:  
NOTE: If a previously ZIKV RNA negative participant has ZIKV-like symptoms at a 
scheduled study visit, follow the ZIKV/DENV laboratory testing algorithm outlined 
in Section 7.1.6, Appendix I and the HIV ZIP MOP. 
– Third trimester only (Do not repeat previously positive tests): Blood for 
TORCH testing and syphilis (RPR) for clinical care and send to local 
laboratory. 
– Blood for ZIKV RNA detection.  
– Blood for ZIKV serology.  
– Blood for DENV serology and other possible serology as needed (CKNV or 
WNV) if resides in or recent travel history to DENV-endemic region. (Do not 
collect if previously tested positive.) 
– Blood for plasma for future studies (if consented for future use). 
– HIV-infected women only: Blood for HIV VL and T-cells only if not 
collected within the prior 12 weeks. (HIV VL testing is required if ZIKV-like 
symptoms present, regardless of testing history.) 
– Urine for ZIKV RNA detection.  
– Urine for possible later testing (if consented for future use). 
 Re-educate about ZIKV prevention and the signs and symptoms of acute ZIKV 
infection and instruct to notify the clinic immediately if experiencing these 
symptoms (or if sex partner or household member is experiencing these 
symptoms).  
 Provide reminder to bring Zika-symptom diary to the next study visit (or at time 
of delivery). 
7.1.4 
Delivery Visit (Live Birth or Fetal Loss) 
 The study team obstetrician and nurse will perform interviews and PEs and 
request laboratory testing as part of standard of care. 

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 The following will be completed within 48 hours after delivery:  
– Obtain results from fetal USs performed since the prior study visit. 
– Zika-symptom diary review.  
– Interim medical and medication history. 
– Interim pregnancy/delivery questionnaire. 
– Other risk factors questionnaire.  
– Obtain results from clinical laboratory tests conducted for clinical care.  
– HIV-infected participants: Abstraction of all interim HIV VLs and T-cells 
results.  
– VS (including blood pressure and temperature). 
– Targeted PE. 
 The following biological samples will be collected within 48 hours of delivery:  
NOTE: If a previously ZIKV RNA negative participant has ZIKV-like symptoms at a 
scheduled study visit, follow the ZIKV/DENV laboratory testing algorithm outlined 
in Section 7.1.6, Appendix I and the HIV ZIP MOP. 
– Blood for ZIKV RNA detection.  
– Blood for ZIKV serology.  
– Blood for DENV serology and other possible serology as needed (CKNV or 
WNV) if resides in or recent travel history to DENV-endemic region. (Do not 
collect if previously tested positive.) 
– Blood for plasma for future studies (if consented for future use). 
– HIV-infected women only: Blood for HIV VL and T-cells only if not 
collected within the prior 12 weeks. (HIV VL testing is required if ZIKV-like 
symptoms present, regardless of testing history. See Section 7.1.6.) 
– Urine for ZIKV RNA detection.  
– Urine for possible later testing (if consented for future use). 
– Cord blood for possible future ZIKV testing (if consented for future use). 
(Some cord blood will be used for real-time testing if infant peripheral blood 
is unobtainable.) 

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– Placental tissue for possible future analysis (if consented for future use). 
 Re-educate about ZIKV prevention and the signs and symptoms of acute ZIKV 
infection and instruct to notify the clinic immediately if experiencing these 
symptoms (or if sex partner or household member is experiencing these 
symptoms).  
 Provide reminder to bring Zika-symptom diary to the next study visit. 
7.1.5 
Six Weeks Postpartum Visit (Post Live Birth or Fetal Loss) 
 The following will be completed at the Six Weeks Postpartum visit:  
– Zika-symptom diary review.  
– Interim medical and medication history. 
– Other risk factors questionnaire.  
– Obtain results from clinical laboratory tests conducted for clinical care.  
– HIV-infected participants: Abstraction of all interim HIV VLs and T-cells 
results.  
– Weight and VS (including blood pressure and temperature). 
– Targeted PE.  
 The following biological samples will be collected:  
– Blood for ZIKV RNA detection.  
– Blood for ZIKV serology.  
– Blood for DENV serology and other possible serology as needed (CKNV or 
WNV) if resides in or recent travel history to DENV-endemic region. (Do not 
collect if previously tested positive.) 
– Blood for plasma for future studies (if consented for future use). 
– HIV-infected women only: Blood for HIV VL and T-cells only if not 
collected within the prior 12 weeks. (HIV VL testing is required if ZIKV-like 
symptoms present, regardless of testing history. See Section 7.1.6.) 
– Urine for ZIKV RNA detection.  
– Urine for possible later testing (if consented for future use). 

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7.1.6 
Women Experiencing Symptoms Consistent with Acute ZIKV-like Illness at Any 
Time during Study Participation 
 If the study staff become aware of a symptomatic participant between study visits, 
schedule an “unplanned symptomatic” visit and conduct the following: 
– Abstract results from fetal US performed according to country and local 
clinical guidelines since the prior study visit. 
– Zika-symptom diary review.  
– Interim pregnancy questionnaire. 
– Continental U.S. sites only: Other risk factors questionnaire. 
– Obtain results from clinical laboratory tests conducted for clinical care.  
– HIV-infected participants: Abstraction of all interim HIV VLs and T-cells 
results.  
– Weight and VS (including blood pressure and temperature). 
– Targeted PE.  
 The following biological samples will be collected:  
– Blood for ZIKV RNA detection.  
– Blood for ZIKV serology.  
– Blood for DENV serology and other possible serology as needed (CKNV or 
WNV) if resides in or recent travel history to DENV-endemic region. (Do not 
collect if previously tested positive.) 
– Blood for plasma for future studies (if consented for future use). 
– HIV-infected participants: Blood for HIV VL. 
– Urine for ZIKV RNA detection. 
– Urine for possible later testing (if consented for future use). 
 Provide reminder to bring Zika-symptom diary to the next study visit (or at time 
of delivery, if applicable). 

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7.1.7 
Women with Positive ZIKV Serology at Any Study Visit, But without Clinical 
Signs/Symptoms of ZIKV-like Illness  
 An additional study visit will not be scheduled and additional biological samples 
will not be collected. 
 Available biological samples will be tested as follows:  
– Serum and urine from current visit for ZIKV RNA detection. 
– Serum and urine from most recent prior visit for ZIKV RNA detection, if 
applicable. 
– If ZIKV RNA test is negative only: Serum for DENV serology and other 
possible serology as needed (CKNV or WNV) if resides in or recent travel 
history to DENV-endemic region.  
 In addition, future specimens collected may be tested for the presence of ZIKV. 
7.2 
Infant Study Visits  
See Appendix II for the schedule of evaluations. Detailed guidelines for specimen 
collection, processing, storage and shipping can be found in the HIV ZIP MOP. 
7.2.1 
Birth Visit 
 The following will be completed within 48 hours after birth: 
– Confirm/review consent and study visit requirements for the infant with 
parent(s)/legal guardian(s) prior to conducting any infant procedures.  
– Collect demographic and birth data.  
– Height, weight, head circumference and temperature. 
– General PE or collect data from exam conducted as part of clinical care. 
– Neurological examination (seizures, apnea, reflexes/tone, asymmetry, eye 
opening, basic movement). 
– Hearing assessment (if abnormal refer to audiology for a full audiological 
examination and obtain results from any tests done for clinical reasons). 
– Ophthalmologic evaluation (if abnormal refer for additional testing and 
obtain results from any tests done for clinical reasons). 
– Obtain results from all neuroimaging assessments done for clinical reasons 
(head US, head CT and MRI scan).  

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– Record results from laboratory tests conducted for clinical care, including 
TORCH testing and HIV-related laboratory testing results and treatments, if 
applicable.  
 The following biological samples will be collected:  
NOTE: Peripheral blood will be collected from the infant, but if unobtainable, some 
of the cord blood will be used for real-time and reflex testing. 
– Blood and/or urine for TORCH testing and syphilis (RPR) to local laboratory, 
if clinically indicated and according to local practice (see the HIV ZIP MOP 
for a table on general guidelines for TORCH and syphilis testing for 
asymptomatic newborn infants according to maternal history). 
– Blood for ZIKV serology and RNA detection.  
– Blood for DENV serology and other possible serology as needed (CKNV or 
WNV). 
– Urine for ZIKV RNA detection. 
– Urine for possible later testing (if consented for future use). 
– Remnant CSF samples for possible future testing, if obtained for clinical care 
(if consented for future use). 
7.2.2 
Months 3, 6 and 12 Visits 
 The following will be completed at 12, 24 and 52 weeks after birth (+/- 2 weeks):  
– Height, weight, head circumference and temperature. 
– General PE or collect data from exam conducted as part of clinical care if 
performed within the previous four weeks. 
– Neurological examination (seizures, apnea, reflexes/tone, asymmetry, eye 
opening, basic movement). 
– Hearing assessment (if abnormal refer to audiology for a full audiological 
examination and obtain results from any tests done for clinical reasons). 
– Ophthalmologic evaluation (if abnormal refer for additional testing and obtain 
results from any tests done for clinical reasons). 
– Obtain results from all neuroimaging assessments done for clinical reasons 
(head US, head CT and MRI scan).  

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– 3 Month visit only: Perform head US only if Zika-exposed and no head US 
was performed by 3 months of age.  
– Record results from laboratory tests conducted for clinical care, including 
TORCH testing and HIV-related laboratory testing results and treatments, if 
applicable.  
 The following biological samples will be collected:  
– Blood for ZIKV serology and RNA detection.  
– Blood for DENV serology and other possible serology as needed (CKNV or 
WNV). 
– Urine for ZIKV RNA detection. 
– Urine for possible later testing (if consented for future use). 
– Remnant CSF samples for possible future testing, if obtained for clinical care 
(if consented for future use). 
8.0 
Specimen Tracking and Storage 
8.1 
Specimen Tracking 
Laboratory specimens processed and shipped to the designated central laboratories for 
testing or to the NICHD Biorepository for storage will not contain names, addresses, 
medical record numbers or any other PII that may be linked to participants. Instead, a 
unique identification number will be used and the types of specimens collected will 
be recorded on CRFs and the information entered into the study database.  
8.2 
Biorepository Storage 
HIV ZIP will also store specimens in the NICHD Biorepository for future studies that 
are currently undetermined.  
Biorepository samples will be processed immediately after collection, labeled, stored 
at the sites in a standardized manner, tracked utilizing the Laboratory Data 
Management System (LDMS) and shipped to the Biorepository on a scheduled basis. 
The LDMS will have the capability of identifying the precise location of every 
Biorepository specimen at all times. Refer to the HIV ZIP LPC for specific storage 
and shipping instructions and schedules. 

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9.0 
Participant and Study Management 
9.1 
Tracking Participants / Follow-up 
After consent (assent with parent(s)/legal guardian(s) permission) is obtained, 
participants will be asked to provide several means to contact them (e.g., home and 
cell phone numbers, mailing address, email, text messaging) including family 
members and friends, if possible and willing to do so. Participants will be asked 
whether or not messages can be left for each of the phone numbers provided and if 
messages can contain information regarding the nature of the project. 
Participants will be contacted by study staff via their preferred methods of contact 
and reminded of follow-up visit dates and times. Site staff should review contact 
information at each study visit with participants to confirm or update information as 
necessary.  
9.2 
Retention  
Participant retention is considered a high priority. Study-wide targets for retention in 
HIV ZIP will be 95% of pregnant women through delivery and 95% of infants 
through 12 months of age, excluding unavoidable causes of loss (e.g., a move out of 
the area or death). The targets will be periodically reevaluated based on ongoing 
experience and efforts made to enhance retention. For those participants who move to 
a new location that has an HIV ZIP site, the originating and receiving site personnel 
will make every effort to encourage continued participation at the new site. Sites will 
be required to submit and have approved a detailed retention plan as part of their site 
implementation plan. 
9.3 
Study Visit Management 
Study visits will be scheduled and conducted as outlined in the schedule of 
evaluations (see Section 7.0 and Appendices I and II). If a visit cannot be conducted 
within the assigned study visit window, site staff should query the HIV ZIP Protocol 
Team using the HIV ZIP QNS, which is accessible through the HIV ZIP link on the 
NICHD Clinical Studies website (www.nichdclinicalstudies.org), requesting 
permission to conduct the visit outside the designated study visit window.  
9.4 
Premature Study Discontinuation  
The HIV ZIP Protocol Team will monitor the rate and reason for discontinuing 
follow-up. Participants will be discontinued from the study if any of the following 
occurs:  
 The participant or parent(s)/legal guardian(s) withdraws consent; 
 The participant or parent(s)/legal guardian(s) fails to comply with the study 
requirements so as to cause harm to the participant or seriously interfere with the 

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validity of the study results and the site investigator believes that compliance is 
unlikely to improve;  
 The site investigator determines that further participation would be detrimental to 
the participant’s health or well-being;  
 The study is stopped by a government agency, including the National Institutes of 
Health (NIH) or U.S. Department of Health and Human Services (HHS); 
 The study must be stopped for administrative reasons; or 
 The clinical site is terminated for participant safety, study integrity or poor 
performance reasons. 
9.5 
Death of a Participant 
Sites will make their best attempt to obtain a copy of the autopsy report or death 
certificate and medical records on any participant who dies.  
10.0 
Data Collection and Management 
10.1 
Development of Protocol and Case Report Forms 
Westat, in collaboration with the HIV ZIP Protocol Team, is responsible for the 
development of this protocol as well as the CRFs needed to collect the information 
required to implement this protocol. All CRFs for this study will be available for 
download from the HIV ZIP link on the NICHD Clinical Studies website 
(www.nichdclinicalstudies.org); hard copies of CRFs may be ordered from that 
website as well.  
10.2 
Data Records  
Participant-related study information will be identified through the PID on all CRFs. 
Participant names or other PII will not be used on any study documents. A log that 
links the names of participants to their PID will also be kept under double locks, 
separate from all other research records. Clinical research records are stored in a 
manner that ensures privacy, confidentiality, security and limited accessibility when 
the clinical research is being conducted and after the research is completed. Records 
can be maintained in hardcopy, electronic or other media form. It is permissible to 
transfer these documents from paper records to electronic formats and to archive this 
information on available media. 
Data collection is the responsibility of the research staff at the sites under the 
supervision of the site investigators. During the study, the site investigators must 
maintain complete and accurate documentation for the study data. Data elements 
transcribed onto CRFs must have corresponding paper or electronic source 
documentation to substantiate all submitted data (e.g., an original or certified copy of 

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a laboratory report, instrument printout, progress notes of the physician, the study 
participant’s hospital chart(s), and nurses’ notes). Research site staff should refer to 
the NIAID Requirements for Source Documentation in the Division of Acquired 
Immunodeficiency Syndrome (DAIDS) Funded and/or Sponsored Clinical Trials (see 
the HIV ZIP MOP). 
10.3 
Data Submission 
Data obtained in the HIV ZIP study will be entered and managed in a 21 Code of 
Federal Regulations (CFR) Part 11-compliant, secure encrypted remote data capture 
(RDC) system called REDCap Cloud. Access to the REDCap Cloud system is limited 
to approved individuals; the PIs and Westat will set the level of access for individuals 
to the database.  
Study site staff must follow the guidelines for CRF completion and data entry that are 
specified in the HIV ZIP MOP. Once the study database is developed, the data 
screens are available for data entry and protocol training has been completed, study 
staff at the sites will be responsible for ensuring that CRF data are entered into the 
study database within the timeframes specified in the HIV ZIP MOP. 
10.4 
Data Quality Assurance 
The site investigators are responsible for ensuring the accuracy, completeness, 
legibility and timeliness of the data reported. All source documents and laboratory 
reports must be reviewed by the site’s study team, who will ensure that they are 
accurate and complete. The investigator or designee must review unanticipated 
problems. Data reported on the CRF that are derived from source documents or chart 
review should be consistent with the source documents or the discrepancies should be 
explained in the participant’s research record. All CRFs must be reviewed by the 
research staff for accuracy, clarity, and completion, and by the data entry staff for 
completion. Data edits through range checks and field inconsistencies will be built 
into the REDCap Cloud database to enable real-time correction of data entry and/or 
CRF completion errors.  
Investigators receiving federal funding must adhere to the CFR to protect research 
participants and produce reliable study information. The PI at each study site is 
responsible for the overall conduct of research activities at the site. Clinical sites 
participating in research sponsored by NICHD need to have an internal quality 
assurance plan that will identify problems and correct errors in research study 
records.  
10.5 
Clinical Site Monitoring and Record Availability 
Site monitors from Westat will visit participating clinical research sites to review 
select study documents (e.g., consent forms, CRFs), pertinent hospital and clinic 
records, as applicable, and ensure that regulatory requirements are being followed. 

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The site investigator will make study documents readily available for inspection by 
the local IRB/IEC, site monitors, NIH, the Office for Human Research Protections 
(OHRP) and the site monitors acting on behalf of NICHD to confirm the study data 
and regulatory compliance. 
NOTE: Participating sites are responsible for specifying these individuals and the 
HIV ZIP investigators as recipients of private health information in the individual’s 
authorization required under the Health Insurance Portability and Accountability Act 
(HIPAA). 
10.6 
Retention of Clinical Research Records 
Records relating to research and IRB/IEC records are required by the HHS 
regulations 45 CFR Part 46.115(b) to be retained for at least three years after 
completion of the research. Participating sites must follow their local institution’s 
policy on record retention if it is for a longer period.  
10.7 
Protocol Query Management 
For the integrity of the study and the welfare of the participants, it is important for the 
site staff to have immediate access to the research team. Site staff will send all queries 
for this protocol to the HIV ZIP Protocol Team using the HIV ZIP QNS accessible 
via the HIV ZIP link on the NICHD Clinical Studies website 
(www.nichdclinicalstudies.org). It is expected that the Protocol Co-Chairs or 
designees will respond to queries within two business days of team receipt. Queries 
and replies will automatically be archived by the HIV ZIP webmaster. Those queries 
deemed relevant to all sites will be posted on the HIV ZIP website, where they will be 
available to all sites for future reference. The categories of queries include 
IRB/Regulatory, Laboratory/Procedures, General Protocol Issues, Participant 
Management/Eligibility/Deviations and Unanticipated Problems Notification. 
10.8 
Protocol Deviations 
A protocol deviation is any noncompliance with the clinical study protocol, Good 
Clinical Practice (GCP) or HIV ZIP MOP requirements. The noncompliance may be 
on the part of the participant, the investigator or study staff. As a result of deviations, 
corrective actions are to be developed by the study staff and implemented promptly. 

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These practices are consistent with investigator and Sponsor obligations in ICH E6 (R1): 
 
 Compliance with Protocol, Sections 4.5.1, 4.5.2, 4.5.3 and 4.5.4. 
 Quality Assurance and Quality Control, Section 5.1.1. 
 Noncompliance, Sections 5.20.1 and 5.20.2. 
Deviations from the protocol must be addressed in the participant’s source documents 
and those that compromise participant safety must be promptly reported to the HIV 
ZIP Protocol Team via the HIV ZIP QNS. Deviations should be reported to the local 
IRB/IEC, according to their requirements.  
10.9 
Collaboration with Outside Studies 
Periodically, it will be useful for the HIV ZIP protocol team to collaborate with 
outside investigators. For example, data from the ZIP study, conducted in Zika-
endemic areas, may be merged with HIV ZIP. In most cases, ZIP will have the exact 
same infant assessments as HIV ZIP. A memorandum of understanding (MOU) about 
the extent and nature of the sharing as well as a data use agreement will be executed 
for all collaborations. The MOU will include an understanding of the control of the 
use of the data, publication rights and authorship rights, as well as address the human 
participant’s confidentiality issues. 
11.0 
Monitoring Unanticipated Problems 
Since this is an observational, and not a treatment or interventional study, AE 
reporting of abnormal medical or laboratory observations at the individual participant 
level are not required.  
Safety monitoring for this study will focus on unanticipated problems involving (1) 
risks to participants; (2) risks to study site staff; and (3) risks to the community. Some 
unanticipated problems involve social or economic harm instead of the physical or 
psychological harm associated with AEs. In other cases, unanticipated problems place 
participants or others at increased risk of harm, but no harm occurs. Women enrolled 
in HIV ZIP may develop known conditions associated with pregnancy that require 
intervention. They may also develop untoward effects possibly associated with ZIKV 
infection. Infants enrolled in HIV ZIP may develop common pediatric conditions 
requiring treatment during the course of the study period. They may also experience 
AEs possibly associated with ZIKV exposure. Although these events will be 
documented and entered in the study database, they will not be categorized as 
unanticipated problems.  
Reporting of unanticipated problems negatively affecting study participants, staff or 
the community will facilitate the reexamination of study procedures and allow 

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changes as necessary to address concerns about participant management, adequacy of 
training or the need to modify procedures.  
11.1 
Unanticipated Problems Definition 
The OHRP considers unanticipated problems involving risks to participants or others 
to include, in general, any incident, experience, or outcome that meets all of the 
following criteria: 
 Unexpected in terms of nature, severity or frequency given (a) the research 
procedures that are described in the protocol-related documents, such as the 
IRB/IEC-approved research protocol and IC document; and (b) the characteristics 
of the patient population being studied; 
 Related or possibly related to participation in the research (possibly related means 
there is a reasonable possibility that the incident, experience or outcome may have 
been caused by the procedures involved in the research); and 
 Suggests that the research places participants or others at a greater risk of harm 
(including physical, psychological, economic or social harm) than was previously 
known or recognized. 
11.2 
Unanticipated Problems Categories 
11.2.1 
Study Participants 
First, the study will catalogue any unanticipated problem experienced by the maternal 
or child participant that may be attributable to participation in or the conduct of the 
study. Reporting is required for occurrences, including social harms, psychological 
distress and serious life threatening events, such as suicide attempts. These may be 
immediately apparent to the study staff, such as the participant’s emotional upset 
requiring referral for counseling; or they may be delayed and reported later to study 
staff, such as physical harm to an individual for having participated in the study.  
11.2.2 
Site Staff 
Second, study staff may encounter unanticipated problems during the study when 
working with the study participants, significant others or extended family members 
that may personally affect them (e.g., visible distress or injury to a staff member 
resulting from the research encounter). Notification to the HIV ZIP Protocol Team 
will allow the problem to be immediately addressed and evaluated, and for guidance 
to be modified or expanded to minimize similar risk to other staff.  
11.2.3 
The Community 
Third, a critically important area that will be evaluated by the HIV ZIP Protocol 
Team is the impact of the study on the community. Community-associated 
unanticipated problems are events that affect the site, site milieu or the performance 

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of the study. This may include events that affect the medical center environment or 
the perception of the study in the community (e.g., the study is portrayed negatively 
in the media or a community forum). Reporting of these events will facilitate 
understanding of the impact of the study on the community and provide the 
opportunity to address community-level concerns and to intervene in a timely manner 
to correct misinformation or perceptions of practices that may cause community 
concern. 
11.3 
Reporting Requirements 
Ultimately, the responsibility for reporting unanticipated problems belongs to the site 
PI who can delegate it to qualified research staff that directly interact with the 
participants. No unanticipated problem should be reported through the HIV ZIP QNS 
without the knowledge of the PI or his or her designee during the PI’s absence. 
Study staff will notify the protocol team of unanticipated problems affecting study 
participants, study staff and the community as soon as possible, but no later than 48 
hours after awareness of the event using the HIV ZIP QNS accessible through the 
HIV ZIP link on the NICHD Clinical Studies website (www.nichdclinicalstudies.org). 
Study staff will also report the unanticipated problem on the HIV ZIP Unanticipated 
Problems Form for entry into the study database within three working days after 
awareness of the event. Lastly, per OHRP, study staff should report unanticipated 
problems to their local IRB/IEC within one week of awareness of the problem if 
serious; otherwise, within two weeks, and to OHRP within one month. 
11.4 
Grading of Impact 
The grading system for unanticipated problems of study participants or staff will be 
as follows: 
 Minimal Impact: managed at the time of the event with no consequences. 
 Moderate Impact: managed by referral for supplemental care or counseling. 
 Major Impact: needed immediate professional intervention with or without 
hospitalization. 
Community events will not be graded but will be addressed as they occur.  
NOTE: The distinguishing feature of moderate and major impact events is the need 
for enlisting additional support outside the research staff and the research encounter. 
 
 

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12.0 
Data Analysis Plan 
The following data analysis plan has been developed to address the study aims. 
12.1 
Aim 1: To Determine the Feasibility of Enrolling Pregnant Women with ZIKV/HIV 
Co-infection, HIV Infection Alone, ZIKV Infection Alone as well as Doubly 
Uninfected Women into a Prospective Cohort Study at Selected Sites in the 
Continental U.S., P.R. and Brazil 
Feasibility in enrolling a total of 200 pregnant women/infant pairs within a year, with 
a target of 150 HIV-infected women across all sites, 50 HIV-uninfected women from 
continental U.S. sites only and a minimum of 20 who are co-infected with HIV and 
ZIKV will be assessed by monitoring total accrual, accrual by study sites and accrual 
into the four study groups described above, at bi-weekly or monthly intervals. 
Monitoring of accrual at consistent study intervals will allow for assessment of trends 
over time and forecasting of accrual over the remaining first year of enrollment. 
If the feasibility phase proves successful, enrollment into Phase II will begin with the 
goal of enrolling up to 1,800 pregnant women/infant pairs at risk for ZIKV.  
12.2 
Aim 2: To Compare HIV Viral Suppression in HIV-Infected Women with and 
without ZIKV Co-infection during Pregnancy and the Time of Delivery 
To address this aim, the study population will be restricted to HIV-infected women. 
Lack of HIV viral suppression (e.g., “unsuppressed VL”) will be defined as having a 
VL > 1,000 copies/ml as this is the VL threshold identified as being associated with 
HIV transmission. HIV viral suppression will be assessed at four time points – start of 
first, second and third trimesters (using the closest VL measure to the start of each 
trimester) and at the time of delivery (closest VL measure prior to or at the time of 
delivery). The proportion with unsuppressed VL at these time points will be 
compared by presence of ZIKV co-infection at these same time points using Fisher’s 
Exact tests. Logistic regression models will be used to estimate ORs and 95% CIs for 
lack of HIV viral suppression at these time points comparing HIV-infected pregnant 
women with ZIKV co-infection to HIV-infected pregnant women without ZIKV co-
infection, adjusting for potential confounders. We hypothesize that HIV-infected 
pregnant women with ZIKV co-infection will have a higher risk of having 
unsuppressed VL during pregnancy and at the time of delivery compared to HIV-
infected pregnant women without ZIKV infection. For this aim and all subsequent 
aims for Phase II, a p-value < 0.05 will connote statistical significance. 
12.3 
Aim 3: To Compare the Incidence of ZIKV Infection among Pregnant Women with 
HIV Infection and Those without HIV Infection 
To address this aim, the study population will be restricted to women without 
prevalent ZIKV infection at the time of enrollment (i.e., those at risk for incident 
ZIKV infection during study follow-up). Cumulative incidence of ZIKV infection 
will be calculated as the number of new confirmed ZIKV infections identified by the 

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end of pregnancy divided by the total number of completed pregnancies, overall and 
by HIV status. The relative risk and 95% CI for ZIKV infection comparing HIV-
infected women to HIV-uninfected women will be estimated using log-binomial 
regression, adjusting for potential confounders.  
12.4 
Aim 4: To Compare the Incidence of Adverse Pregnancy Outcomes between Women 
Co-infected with HIV and ZIKV, Women Infected with Either HIV or ZIKV Alone 
and Doubly Uninfected Women 
The cumulative incidence of adverse pregnancy outcomes (i.e., miscarriage, stillbirth 
and preterm birth) will be calculated as the number of adverse outcomes identified by 
the end of pregnancy divided by the total number of completed pregnancies, overall 
and by HIV/ZIKV status. The relative risk and 95% CI for adverse pregnancy 
outcome comparing HIV and ZIKV co-infected, HIV-infected only and ZIKV-
infected only women to doubly uninfected women will be estimated using log-
binomial regression, adjusting for potential confounders. Potential confounders 
include demographic and socioeconomic status variables, smoking, alcoholic 
beverage consumption and substance use during pregnancy and exposure to specific 
environmental contaminants during pregnancy.  
12.5 
Aim 5: To Compare the Incidence of Vertical Transmission of HIV and ZIKV 
between Women Co-infected with HIV and ZIKV, and Women Infected with Either 
HIV or ZIKV Alone 
To address this aim, the study population will be restricted to women who are 
infected with HIV, ZIKV or both. Among the doubly infected pregnant women, we 
will estimate the cumulative incidence of perinatal transmission of both HIV and 
ZIKV, HIV only and ZIKV only. Among HIV-infected only pregnant women, we 
will estimate the risk of perinatal HIV transmission. Among ZIKV-infected only 
pregnant women, we will estimate the risk of perinatal ZIKV transmission. We will 
compare the risk of perinatal HIV transmission among doubly infected women to 
women only infected with HIV using log-binomial regression, adjusting for potential 
confounders. Similarly, we will compare the risk of perinatal ZIKV transmission 
among doubly infected women to women only infected with ZIKV using log-
binomial regression, adjusted for potential confounders.  
12.6 
Aim 6: To Compare the Incidence of Congenital Malformations and Other Adverse 
Outcomes (Including Microcephaly, Neonatal Death, CNS Malformations, Hydrops 
and Ocular Abnormalities) among Offspring of Women Co-infected with HIV and 
ZIKV, Women Infected with Either HIV or ZIKV Alone and Doubly Uninfected 
Women 
The cumulative incidence of congenital malformations and other adverse outcomes 
among infants will be calculated overall and by perinatal HIV/ZIKV exposure status. 
The risk ratios and 95% CI for adverse infant outcomes comparing HIV and ZIKV 
co-infected, HIV-infected only and ZIKV-infected only women to doubly uninfected 
women will be estimated using log-binomial regression, adjusting for potential 

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confounders. Potential confounders include demographic and socioeconomic status 
variables, substance use during pregnancy and exposure to specific environmental 
contaminants during pregnancy.  
12.7 
Aim 7: To Compare Long-term Growth, Hearing, Vision and Neurodevelopment 
Outcomes in Children with In Utero Exposure to HIV and ZIKV, In Utero Exposure 
to Either HIV or ZIKV Alone and No In Utero Exposure to Either Virus  
The incidence rate of long-term outcomes among infants will be calculated as the 
number of adverse outcomes identified over follow-up divided by the total person-
time of follow-up, overall and by perinatal HIV/ZIKV exposure status. The incidence 
rate ratios and 95% CI for adverse infant outcomes comparing doubly exposed, HIV-
exposed only and ZIKV-exposed only women to doubly uninfected women will be 
estimated using Poisson regression, adjusting for potential confounders. Potential 
confounders include demographic or socioeconomic status variables, substance use 
during pregnancy and exposure to specific environmental contaminants during 
pregnancy.  
12.8 
Missing, Unused and Spurious Data 
Every effort will be made to ensure that the amount of missing data is kept at a 
minimum. The extent and pattern of missing data will be assessed. If data are missing 
for only a few cases, then data analysis will be conducted using the standard complete 
case analyses as outlined above. However, when such a strategy would result in loss 
of data from a substantial proportion of participants or lead to biased or inaccurate 
results, then multiple imputation or inverse probability weighting will be considered.  
Unused or spurious data will be documented and discussed when disseminating 
results of this study. 
12.9  
Power and Sample Size 
Although the key goal of Phase I is to assess feasibility of enrolling HIV-infected and 
HIV-uninfected women with or without ZIKV infection, it should be noted that the 
proposed sample size of 150 HIV-infected women would provide 80% power to 
detect a difference in percent with unsuppressed VL of 38.7% vs. 10% between HIV-
infected women with ZIKV and HIV-infected women without ZIKV infection, 
assuming 10% of the HIV-infected women enrolled (and meeting ZIKV risk criteria) 
have ZIKV infection by delivery. 
In the full Phase II study, we anticipate enrolling an additional 1800 pregnant women 
to bring total enrollment for this study up to 2000 women. This large enrollment 
allows precise estimates of event rates and sufficient power to compare subgroups 
defined on the basis of prenatal HIV/ZIKV exposure or other characteristics. We will 
first present estimates of precision for various event rates for the full cohort.  

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Assuming an enrollment of 500, 1,000, or 2,000 women, Table 12-1 below 
summarizes estimates of precision of event rates for the development of ZIKV by the 
end of pregnancy, or for other adverse outcomes in their infants. For the target 
enrollment of 2,000 women, we will be able to estimate an overall event rate of 10% 
with a precision (i.e., 1.96* s.e.) of ± 1.4%. If 1,000 of these 2,000 women are HIV-
infected, then we will have a precision of 1.8% for estimating the percent with ZIKV 
infection within this subgroup. 
Table 12-1. 
Precision of event rate estimates for various levels of accrual, overall or within 
subgroups defined by HIV status 
Accrual (overall or within 
HIV+ or HIV- subgroups) 
Percent with ZIKV infection by end of pregnancy,  
or other adverse outcome in infants 
Event rate 
Precision 
(half-width of 95% CI) 
500 
5% 
2.0% 
 
10% 
2.8% 
 
20% 
3.6% 
1000 
5% 
1.4% 
 
10% 
1.9% 
 
20% 
2.5% 
2000 
5% 
1.0% 
 
10% 
1.3% 
 
20% 
1.8% 
 
For comparison of the percent with unsuppressed VL in Aim 2, our power is based 
only on the HIV-infected women enrolled. Assuming that a range of 40% to 60% of 
the total enrollment is HIV-infected, this translates to 200 to 1,200 women with HIV 
infection for corresponding total enrollments of 500 to 2,000 participants. Table 12-2 
below summarizes the minimum detectable differences in proportions with 
suppressed VL by the end of pregnancy (and corresponding ORs) between HIV-
infected ZIKV-exposed as compared to HIV-infected ZIKV-unexposed women. 
 
 

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Table 12-2. 
Minimum detectable ORs (and percent with unsuppressed VL among ZIKV-
infected) at 80% power comparing HIV+/ZIKV+ vs. HIV+/ZIKV- women 
Assumed percent 
with unsuppressed 
VL in 
ZIKV-uninfected 
Percent 
ZIKV-infected 
(among HIV-
infected 
enrollees) 
Minimum detectable ORs (percent with 
unsuppressed VL in ZIKV+) for sample sizes: 
N=200 
N=500 
N=1200 
10% 
20% 
3.54 (28.2%) 
2.36 (20.8%) 
1.80 (16.7%) 
 
30% 
3.16 (26.0%) 
2.17 (19.4%) 
1.69 (15.8%) 
 
40% 
3.01 (25.1%) 
2.09 (18.9%) 
1.65 (15.5%) 
 
50% 
2.99 (25.0%) 
2.08 (18.8%) 
1.64 (15.4%) 
 
60% 
3.09 (25.6%) 
2.12 (19.1%) 
1.66 (15.5%) 
 
70% 
3.34 (27.1%) 
2.24 (19.9%) 
1.72 (16.0%) 
20% 
20% 
2.89 (42.0%) 
2.01 (33.5%) 
1.60 (28.5%) 
 
30% 
2.58 (39.2%) 
1.86 (31.8%) 
1.51 (27.4%) 
 
40% 
2.46 (38.1%) 
1.80 (31.1%) 
1.48 (27.0%) 
 
50% 
2.44 (37.9%) 
1.79 (30.9%) 
1.47 (26.8%) 
 
60% 
2.49 (38.4%) 
1.81 (31.2%) 
1.48 (27.0%) 
 
70% 
2.65 (39.9%) 
1.89 (32.1%) 
1.52 (27.6%) 
 
For example, at the full target sample size of 2,000 women, assuming 60% (N=1200) 
are HIV-infected, the study design provides 80% power to detect a difference in the 
percent with unsuppressed VL of 16.7% vs. 10% comparing ZIKV-infected vs. 
ZIKV-uninfected women, assuming that 20% of the 1200 become ZIKV-infected by 
the end of their pregnancy, corresponding to an OR equal to 1.80. The range of 10% 
to 20% assumed for percent with unsuppressed VL was based on data from the 
PHACS SMARTT study on HIV-infected but presumed ZIKV-uninfected women.[92] 
If the percent with ZIKV infection increases to 50%, the minimum detectable OR at 
80% power decreases to 1.64 reflecting a difference between 15.4% vs. 10%, or an 
absolute difference of 5.4% in unsuppressed VL. Higher rates of unsuppressed VL 
(20%) among the ZIKV-uninfected will also translate to smaller detectable ORs, with 
absolute differences in percent with unsuppressed VL ranging from 7% to 9%. 
For Aim 3, we provide the power for detecting differences in the percent with ZIKV 
infection by the end of pregnancy between HIV-infected and HIV-uninfected women, 
based on interim enrollment of 500, 1,000, and 1,500 women and a final target 
enrollment of 2,000 women, and assuming the percent with HIV infection among 
those enrolled ranges from 40% to 60%. 
 
 

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Table 12-3. 
Minimum detectable ORs (and percent of ZIKV-infected among HIV-infected) at 
80% power for various sample sizes and assumed ZIKV infection rates among 
HIV-uninfected 
Percent of 
enrolled 
with HIV 
infection 
% with 
ZIKV 
infection in 
the HIV-
uninfected 
group 
Minimum detectable ORs  
(percent ZIKV+ in HIV-infected) for sample sizes 
N=500 
N=1000 
N=1500 
N=2000 
40% 
20% 
1.80 (31.1%) 
1.53 (27.7%) 
1.42 (26.2%) 
1.36 (25.3%) 
 
30% 
1.71 (42.2%) 
1.46 (38.6%) 
1.37 (36.9%) 
1.31 (36.0%) 
 
40% 
1.67 (52.7%) 
1.44 (49.0%) 
1.35 (47.3%) 
1.30 (46.3%) 
 
50% 
1.68 (62.7%) 
1.44 (59.0%) 
1.35 (57.4%) 
1.29 (56.4%) 
 
60% 
1.72 (72.1%) 
1.46 (68.7%) 
1.36 (67.1%) 
1.30 (66.2%) 
 
70% 
1.83 (81.0% 
1.52 (78.0%) 
1.40 (76.5%) 
1.33 (75.7%) 
50% 
20% 
1.79 (30.9%) 
1.52 (27.5%) 
1.41 (26.1%) 
1.35 (25.2%) 
 
30% 
1.69 (42.0%) 
1.45 (38.4%) 
1.36 (36.8%) 
1.31 (36.0%) 
 
40% 
1.66 (52.5%) 
1.43 (48.8%) 
1.34 (47.2%) 
1.29 (46.2%) 
 
50% 
1.66 (62.4%) 
1.43 (58.8%) 
1.34 (57.2%) 
1.29 (56.2%) 
 
60% 
1.70 (72.8%) 
1.45 (68.5%) 
1.35 (67.0%) 
1.30 (66.0%) 
 
70% 
1.80 (80.8%) 
1.50 (77.8%) 
1.39 (76.4%) 
1.33 (75.6%) 
60% 
20% 
1.81 (31.2%) 
1.54 (27.7%) 
1.42 (26.2%) 
1.36 (25.4%) 
 
30% 
1.71 (42.3%) 
1.47 (38.6%) 
1.37 (37.0%) 
1.31 (36.0%) 
 
40% 
1.67 (52.7%) 
1.44 (49.0%) 
1.35 (47.3%) 
1.30 (46.3%) 
 
50% 
1.68 (62.6%) 
1.44 (59.0%) 
1.35 (57.4%) 
1.29 (56.4%) 
 
60% 
1.72 (72.0%) 
1.46 (68.6%) 
1.36 (67.1%) 
1.30 (66.2%) 
 
70% 
1.82 (80.9%) 
1.51 (77.9%) 
1.40 (76.5%) 
1.33 (75.7%) 
 
For an example of the interpretation of the results in Table 12-3 above, if 60% of the 
final target of N=2,000 women are HIV-infected and 40% are uninfected, and 
assuming 40% of the HIV-uninfected women become ZIKV-infected by the end of 
pregnancy, then the study design will provide 80% power to detect a difference in 
ZIKV infection of 46.3% vs. 40% for HIV-infected vs. HIV-uninfected women, or an 
absolute difference of 6.3% (with corresponding OR equal to 1.30); at an interim 
enrollment of 1000, the detectable OR would be 1.44 allowing differences in percent 
with ZIKV infection of 9.0% or greater to be detected. 
To address the power for Aim 4, a number of scenarios for possible percentages of 
enrollment within the four different subgroups defined by the cross-classification of 
HIV infection and ZIKV infection were evaluated and the power for comparing the 
percent of pregnancies with adverse outcomes for each of the first three subgroups to 
the doubly uninfected group was evaluated for a range of sample sizes. 

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Table 12-4. 
Power for detecting differences in rate of adverse pregnancy outcomes (for Aims 
4 and 6) based on assumed proportion in the HIV and ZIKV co-infected, HIV-
infected and ZIKV-infected groups as compared to the HIV and ZIKV-uninfected 
group 
Percent of 
enrolled in 
HIV+/ZIKV+, 
HIV+/ZIKV-, 
HIV-/ZIKV+, 
& HIV-/ZIKV- 
groups 
Assumed % 
with adverse 
outcomes in 
HIV-/ZIKV- 
group 
Power for detecting 2-fold difference in odds of adverse outcomes for 
HIV+/ZIKV+, HIV+/ZIKV-, and HIV-/ZIKV+ groups vs. HIV-/ZIKV- 
group 
N=500 
N=1000 
N=1500 
N=2000 
12%, 28%, 
12%, 48% 
5% 
22.2%, 32.8%, 
22.2% 
39.2%, 57.2%, 
39.2% 
54.2%, 74.6%, 
54.2% 
66.4%, 85.7%, 
66.4% 
10% 
36.3%, 54.1%, 
36.3% 
62.4%, 83.1%, 
62.4% 
79.6%, 94.7%, 
79.6% 
89.6%, 98.5%, 
89.6% 
20% 
54.5%, 77.0%, 
54.5% 
83.4%, 96.8%, 
83.4% 
94.8%, 99.7%, 
94.8% 
98.5%, 100%, 
98.5% 
20%, 20%, 
24%, 36% 
5% 
25.4%, 25.4%, 
27.1% 
45.0%, 45.0%, 
47.9% 
61.3%, 61.3%, 
64.7% 
73.7%, 73.7%, 
77.0% 
10% 
42.3%, 42.3%, 
45.3% 
70.5%, 70.5%, 
74.0% 
86.4%, 86.4%, 
89.1% 
94.2%, 94.2%, 
95.8% 
20% 
63.6%, 63.6%, 
67.6% 
90.4%, 90.4%, 
92.8% 
97.9%, 97.9%, 
98.7% 
99.6%, 99.6%, 
99.8% 
15%, 35%, 
10%, 40% 
5% 
23.5%, 32.1%, 
19.3% 
41.6%, 56.1%, 
33.7% 
57.2%, 73.6%, 
47.0% 
69.5%, 84.9%, 
58.6% 
10% 
38.8%, 53.5%, 
31.2% 
65.9%, 82.5%, 
54.7% 
82.7%, 94.4%, 
72.1% 
91.8%, 98.4%, 
83.6% 
20% 
58.4%, 77.2%, 
47.3% 
86.7%, 96.9%, 
76.3% 
96.4%, 99.7%, 
90.6% 
99.1%, 100%, 
96.6% 
25%, 25%, 
20%, 30% 
5% 
25.0%, 25.0%, 
27.1% 
44.3%, 44.3%, 
47.9% 
60.4%, 60.4%, 
64.7% 
72.9%, 72.9%, 
69.3% 
10% 
42.0%, 42.0%, 
45.3% 
70.0%, 70.0%, 
74.0% 
86.1%, 86.1%, 
89.1% 
94.0%, 94.0%, 
92.0% 
20% 
64.0%, 64.0%, 
67.6% 
90.6%, 90.6%, 
92.8% 
98.0%, 98.0%, 
98.7% 
99.6%, 99.6%, 
99.3% 
18%, 42%, 
8%, 32% 
5% 
23.2%, 29.3%, 
16.4% 
41.1%, 51.6%, 
28.0% 
56.5%, 68.8%, 
39.2% 
68.9%, 80.8%, 
49.5% 
10% 
38.6%, 49.4%, 
26.0% 
65.6%, 78.6%, 
46.0% 
82.5%, 92.1%, 
62.4% 
91.6%, 97.3%, 
74.8% 
20% 
58.9%, 73.7%, 
39.4% 
87.0%, 95.6%, 
66.7% 
96.6%, 99.4%, 
83.4% 
99.2%, 99.9%, 
92.3% 
30%, 30%, 
16%, 24% 
5% 
23.0%, 23.0%, 
19.6% 
40.6%, 40.6%, 
34.4% 
55.9%, 55.9%, 
47.9% 
68.3%, 68.3%, 
59.6% 
10% 
38.8%, 38.8%, 
32.4% 
65.8%, 65.8%, 
56.6% 
82.6%, 82.6%, 
74.0% 
91.8%, 91.8%, 
85.2% 
20% 
60.7%, 60.7%, 
50.5% 
88.4%, 88.4%, 
79.7% 
97.1%, 97.1%, 
92.8% 
99.4%, 99.4%, 
97.7% 
 

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From Table 12-4 above, it is evident that the power for detecting an OR of 2 when 
comparing pairs of subgroups (e.g., HIV+/ZIKV+ vs. HIV-/ZIKV-) is low when the 
overall study accrual is 500 or 1,000, but becomes reasonable (> 80%) when the 
background rate of AEs is at least 10% in the doubly uninfected group and the sample 
size is at least 1500, or when the background rate is 20% and the sample size is at 
least 1,000. The first two scenarios in Table 12-4 above reflect an assumption that a 
total of 40% of the women enrolled will be HIV-infected, the second two scenarios 
assume 50% will be HIV-infected, and the last two scenarios assume 60% will be 
HIV-infected. 
Aim 5 involves comparing the rate of transmission of either HIV or ZIKV from 
mother to child. This study is not powered to detect differences in the rate of perinatal 
HIV transmission, but will provide good precision for the estimation of such rates. 
Assuming 40% of women enrolled are HIV-infected, the precision (half-width) of CIs 
will range from 0.055% (for N=2,000) to 0.09% (for N=1,000) under the assumption 
of a 0.5% HIV transmission rate. If 60% of women are HIV-infected, the precision 
would increase and CI half widths would correspondingly decrease to 0.045% 
(N=2,000) to 0.07% (N=1,000). 
The power calculations for Aim 6 are already provided in Table 12-4 for a range of 
AE rates in the doubly uninfected group, and under various scenarios for both interim 
and final study accrual in Phase II, and for percent of total accrual in each of the four 
subgroups. As an example, if the underlying percent with congenital malformations 
or other defects is 10% in the doubly infected group, a sample size of 1,500 will 
generally provide over 80% power for most pairwise comparisons of interest. 
Given the lack of information regarding long-term effects of ZIKV infection in any 
population, and among HIV-infected women in particular, Aim 7 is considered 
exploratory and no power calculations are provided for this specific aim. However, 
the power calculations in Table 12-4 can serve as guidance regarding the range of 
power that would be anticipated for the full study accrual. 
12.10 
Interim Monitoring 
12.10.1 
Accrual and Administrative Monitoring 
During the first year of the study, the enrollment of pregnant women and children into 
the HIV ZIP study will be monitored closely. Total accrual reports will be prepared 
by Westat and distributed on a weekly basis. Monthly accrual reports by site will be 
monitored by the protocol team. Quarterly administrative reports will also be 
prepared, which will summarize baseline characteristics of the cohort along with 
completeness of baseline data, particularly key exposure data such as ZIKV IgM and 
RNA test results. 

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13.0 
Human Subjects 
The study will be conducted in compliance with the protocol, in accordance with the 
ICH guidelines for GCP (ICH E6 (R1)), the CFR on the Protection of Human 
Subjects (45 CFR Part 46), the Belmont Report: Ethical Principles and Guidelines for 
the Protection of Human Subjects of Research and the NIH Office of Extramural 
Research, Research Involving Human Subjects Policies and Guidance.  
13.1 
45 CFR Part 46.404 
It is the judgment of the protocol team that the HIV ZIP protocol belongs in Category 
One Research under 45 CFR Part 46.404: Research not involving greater than 
minimal risk. This judgment is premised on the definition of minimal risk found at 45 
CFR Part 46.102(i): 
Minimal risk means that the probability and magnitude of harm or 
discomfort anticipated in the research are not greater in and of 
themselves than those ordinarily encountered in daily life or during the 
performance of routine physical or psychological examinations or 
tests.  
The principal risks in this study result from the disclosure of sensitive maternal 
information and the risks inherent in the routine examinations that the mother and 
children will receive. These are discussed in more detail in the following sections but 
in essence are medical and psychological examinations employed in routine clinical 
evaluation. 
13.2 
Institutional Review Board / Institutional Ethics Committee and Informed Consent 
This protocol, the IC/assent/parent(s)-legal guardian(s) permission documents (see 
Appendices IV and V), and any subsequent modifications must be reviewed and 
approved by the IRB or IEC responsible for oversight of the study prior to 
implementation. Written IC must be obtained from the participant (or parent(s) or 
legal guardian(s) of participants who cannot consent for themselves, such as those 
below the legal age per state or country regulations). The participant’s assent must 
also be obtained if below legal age and she is able to understand the nature, 
significance and risks of the study. The IC will describe the purpose of the study, the 
procedures to be followed and the risks and benefits of participation. A copy of the 
consent form will be given to the participant (or parent(s) or legal guardian(s)). 
13.3 
Prisoner Participation 
The NICHD concluded that this protocol does not meet Federal requirements 
governing recruitment of prisoners for participation in research and should NOT be 
considered by any local IRB/IEC for this purpose. Participants themselves recruited 
from the general population, who, subsequent to enrollment, become incarcerated or 
are placed in detention, may not continue study participation while incarcerated. 

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13.4 
Participant Confidentiality 
Only a coded number to maintain participant confidentiality will identify all study-
specific laboratory specimens, evaluation forms, reports and other records. All 
records will be kept in a secured area with limited access. A list linking the 
participant names with their assigned coded number will be securely stored at the 
clinical site under double locks, separate from all other research records. All 
electronic research records will be password protected, securely stored and backed 
up. All computer entry and networking programs will be done with coded numbers 
only. Clinical information will not be released without written permission of the 
participant, except as necessary for monitoring by OHRP, the local IRB or IEC, local 
or national regulatory agencies, NICHD, study staff, study monitors and other 
Sponsors, as applicable. 
The Protocol Co-Chairs and all employees and coworkers involved with this study 
may not disclose or use for any purpose other than performance of the study, any 
data, record or other unpublished confidential information disclosed to those 
individuals for the purpose of the study. All research staff persons at the clinical sites 
are required to sign non-disclosure forms pledging to hold research information in 
confidence. Prior written agreement from NICHD must be obtained for the disclosure 
of any said confidential information to other parties. 
All protocol team investigators and collaborators are required to sign data use 
agreements pledging not to seek the identity of study participants.  
PII will not be released by the sites without written permission of the participant, 
except as necessary for monitoring by Westat or NICHD.  
The site investigator will make study documents (e.g., consent forms, CRFs) and 
pertinent records available for inspection by the local IRB or IEC and site study 
monitors on behalf of NICHD, NIH, OHRP or the Sponsor’s designee for 
confirmation of the study data. 
13.5 
Risks and Benefits 
The HIV ZIP Protocol Team has determined that participation in this study involves 
procedures or interventions that are not greater than minimal risk and present the 
prospect of direct benefit to the individual participants. 
The measurements that are involved in this study require venipuncture to collect 
blood samples. This procedure may cause local discomfort, bleeding or bruising; 
rarely can small clot or infection occur at the blood draw site. This measurement 
should not be considered greater than minimal risk in and of itself given its routine 
use in general health care delivery.  
There is some risk that answering questions about some of the topics may be 
uncomfortable or upsetting, such as substance use behaviors. In the event of 

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discomfort or upset, site staff will be able to refer study participants to counseling. 
Participants do not have to answer any question that they do not want to answer. 
Furthermore, participants will be informed that at any point, they may stop if they do 
not wish to continue responding to the questionnaire(s). Every effort will be made to 
keep the participants’ personal information private and confidential, but absolute 
confidentiality cannot be guaranteed. 
There is no guarantee of direct benefit to infants who participate in this study. If 
parents or legal representatives choose, the clinically relevant information obtained in 
this study can be made available to their children’s health care providers and may 
inform their health care. Parents or legal representatives of study participants will be 
encouraged to do this in order to maximize the potential for benefits. 
There may be direct benefit to the women and their infants who participate in the 
study by the testing for ZIKV and other infections at multiple times throughout the 
pregnancy, which may allow for available treatment and management, as well as the 
ability to make choices about the pregnancy. Infants may benefit from the close 
monitoring and frequent evaluations, but there is no guarantee. In addition, future 
pregnant women and their infants may benefit from knowledge that is gained by their 
participation. 
13.5.1 
HIV ZIP Abnormality-Triggered Evaluations 
Infants followed in HIV ZIP will be screened for abnormalities using the tests 
described previously. Abnormal values on these screening measures will “trigger” 
more extensive clinical evaluations, the results of which will be abstracted for the 
HIV ZIP study.  
13.6 
Biorepository Policies 
Biological specimens will be collected and stored for future testing that is not 
specified in the protocol (biorepository specimens). IC (or assent with parent(s)/legal 
guardian(s) permission) will be obtained from individuals as required by 45 CFR Part 
46.116 to have their and their infants’ specimens included in the biorepository.  
Refer to the HIV ZIP MOP for the NICHD Biorepository Policy, April 16, 2010 for 
additional information on the policy addressing the management of biorepository 
specimens, i.e., specimens collected for unspecified future testing and long-term 
storage. 
13.7 
45 CFR § 160 and 164 Standards for Privacy of Individually Identifiable Health 
Information (“Privacy Rule” Pursuant to the HIPAA) 
Each site is responsible for adherence to their individual institution’s HIPAA policies 
and procedures. Sites should ensure that their institutional HIPAA authorization form 
permits this release of protected health information to the clinical provider. 

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13.8 
Study Discontinuation 
The study may be discontinued at any time by NICHD, OHRP, the local IRB or IEC, 
other governmental agencies or local or national agencies, as part of their duties to 
ensure that research participants are protected. 
14.0 
Publication of Research Findings 
This study will comply with the NIH Public Access Policy, which ensures that the 
public has access to the published results of NIH funded research. It requires 
scientists to submit final peer-reviewed journal manuscripts that arise from NIH funds 
to the digital archive PubMed Central upon acceptance for publication.  
Publication of results of this trial will be governed by NICHD policies. Any 
presentation, abstract or manuscript will be made available for review prior to 
submission.  
15.0 
Biohazard Containment 
As the transmission of HIV and other blood-borne pathogens can occur through 
contact with contaminated needles, blood and blood products, appropriate blood and 
secretion precautions will be employed by all personnel in the drawing of blood and 
shipping and handling of all specimens for this study, as currently recommended by 
CDC. These procedures can be found at www.cdc.gov.  
All infectious specimens will be transported in compliance with federal regulations 
and the International Air Transport Association (IATA) Dangerous Goods 
Regulations-Packing Instruction 602. Refer to individual carrier guidelines (e.g., 
Federal Express or World Courier) for specific instructions and to the Guidelines for 
Shipment and Receipt of Category B Biological Substance Shipment and Instruction 
for Overnight Shipments documents (see HIV ZIP LPC).  
All participating sites are also required to follow the specimen management 
procedures outlined in the HIV ZIP MOP for collecting, processing, shipping and 
storing biological specimens for the study. In addition, sites are required to follow 
protocol-specific procedures outlined in the protocol.  
 
 

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Appendix I 
Maternal Schedule of Evaluations for Asymptomatic and Symptomatic Women  
Evaluations / Procedures 
All 
Participants 
 
Enrollment 
Acute 
ZIKV-like 
Symptoms /   
≥ 18 weeks 
gestation 
 
< 18 weeks 
gestation7 
Second 
Trimester  
24 weeks 
gestation  
(+/- 2 weeks) 
Third 
Trimester  
34 weeks 
gestation  
(+/- 2 weeks) 
Delivery  
(live birth or 
fetal loss) 
(+ 48 hours) 
 
6 weeks  
postpartum  
(+/- 7 days) 
On Study / 
Acute ZIKV-
like 
Symptoms 
11  
 
Screening 
Entry 
Entry  
All participants on study 
 
 
 
 
IC 1 
X 2 
X 
X 
 
 
 
 
 
Basic demographics, LMP, eligibility 
confirmation 
X 2 
 
X 
 
 
 
 
 
Fetal US with fetal heartbeat  
X 3 
X 3 
X 3 
X 3 
X 3 
X 3 
 
X 12 
Provide/review Zika-symptom diary 
 
X 
X 
X 
X 
X 
X 
X 
Zika-like symptoms, health & medication history 
 
X 
X 
X 
X 
X 
X 
X 
Pregnancy questionnaire 
 
X 
X 
X 
X 
X 
 
X   
Other risk factors questionnaire 
 
X 
X 
X 
X 
X 
X 
X 13 
Targeted PE 
 
X 
X 
X 
X 
X 
X 
X 
HIV-infected only: HIV VL, absolute CD4/CD8 
T-cells and % abstraction 4 
 
X 
X 
X 
X 
X 
X 
X 
 
LABORATORY EVALUATIONS # 
(Blood volumes in parentheses are collected only if indicated.)  
Urine or blood: βhCG – abstract from medical 
records, if available  
(1 ml) 5 
 
(1 ml) 5 
 
 
 
 
 
Blood: 
        ZIKV IgM Abs  
            ZIKV RNA detection 
        DENV IgM Abs 
 
6 ml 6 
 
6 ml  
6 ml 
 6 ml 
6 ml 
6 ml 
6 ml 
Urine: ZIKV RNA detection 
X 6 
 
X 
(X) 
(X) 
(X) 
(X) 
X 
Blood: TORCH & Syphilis (RPR) blood  8 
 
(3 ml) 
(3 ml) 
 
(3 ml) 
 
 
(3 ml) 
Blood (HIV-infected only): HIV VL 9 
 
(3 ml) 
(3 ml) 
(3 ml) 
(3 ml) 
(3 ml) 
(3 ml) 
3 ml 
Blood (HIV-infected only): CD4/CD8 T-cells 9 
 
(3 ml) 
(3 ml) 
(3 ml) 
(3 ml) 
(3 ml) 
(3 ml) 
 
 
BIOREPOSITORY SPECIMENS (if consented for future use) 
 
Urine  
X 6 
 
X 
X 
X 
X 
X 
X 
Blood for plasma  
(4 ml) 6 
 
(4 ml) 
(4 ml) 
(4 ml) 
(4 ml) 
(4 ml) 
(4 ml) 
Cord blood 10 
 
 
 
 
 
20 ml 
 
 
Placental tissue 
 
 
 
 
 
X 
 
 
TOTAL BLOOD VOLUMES                       
(max. volume per visit = 20 ml) 
Min 0 ml  
Max 11 ml 
Min 0 ml  
Max 9 ml 
Min 9 ml  
Max 20 ml 
Min 6 ml 
Max 16 ml 
Min 6 ml 
Max 19 ml 
Min 6 ml 
Max 16 ml 
Min 6 ml 
Max 16 ml 
Min 9 ml 
Max 16 ml 

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Footnotes: 
#If insufficient blood volume, prioritize as follows: (1) ZIKV IgM Abs and RNA detection; (2) TORCH testing, if not done for clinical care; (3) plasma 
for Biorepository. 
 
1. 
Written maternal IC (or assent with parent(s)/legal guardian(s) permission) must be obtained prior to performing any screening evaluations. Written IC for participation 
of the infant(s) born from this pregnancy may be obtained at the same time or any time during the pregnancy and confirmed at delivery per local requirements. 
2. 
Perform at the Screening visit for all participants.  
3. 
Perform at the Screening or Entry visit ONLY if not done through routine prenatal care. Otherwise, abstract results from all fetal USs performed during prenatal care.  
4. 
At Entry, abstract all available results starting from the date closest, but prior, to the LMP. At all other visits, abstract all available results since the previous visit.  
5. 
Not required if pregnancy already confirmed by fetal US with fetal heartbeat or by βhCG at routine prenatal care.  
6. 
Perform at the Screening visit ONLY for participants 18 weeks or greater GA that present symptomatic for possible acute ZIKV:   
a. 
If ZIKV RNA test is positive, some remaining serum will be tested for ZIKV IgM Abs, the participant will be enrolled in the study and the remaining Entry 
visit evaluations will be performed. 
i. After the Entry visit, the next scheduled study visit will be the one closest to, but occurring after the participant’s current gestation.  
ii. The participant will then follow the same visit schedule as those enrolled at < 18 weeks gestation. See the HIV ZIP LPC for subsequent study visits’ 
testing algorithms based on testing results. 
b. 
If ZIKV RNA test is negative, the participant will not be enrolled in the study; no further testing will be performed and the remaining specimens will be 
destroyed. 
7. 
Participants < 18 weeks gestation that present symptomatic for possible acute ZIKV will be enrolled (if otherwise screened eligible) and have their Entry visit on the 
same day as the Screening visit. Ideally, asymptomatic participants < 18 weeks gestation should be enrolled and have their Entry visit performed on the same day as the 
Screening visit, but they may complete the visit any time prior to 18 weeks gestation. See the HIV ZIP MOP for Entry and subsequent study visit testing algorithms.  
8. 
Abstract results of tests performed for routine prenatal care. Any tests already performed for routine prenatal care do not need to be repeated.  
9. 
Perform testing only if no results are available from within the prior 12 weeks. Perform HIV VL testing if the participant presents symptomatic for possible acute ZIKV, 
regardless of when the last HIV VL was collected. 
10. If peripheral blood is unobtainable from the newborn, send required volume of cord blood to the central laboratory following the Appendix II Birth Visit Laboratory 
Evaluations. Remaining cord blood will go to the Biorepository (if consented for future use). 
11. Follow the “On Study / Acute ZIKV-like Symptoms laboratory collection / testing algorithm for any previously ZIKV RNA negative participant that presents with 
ZIKV-like symptoms while on study. If the study staff become aware of a participant that is newly symptomatic between study visits, schedule an “unplanned 
symptomatic” visit.  
a. 
Collect blood for ZIKV IgM Abs, DENV IgM Abs (unless previously DENV IgM positive), other possible serology and blood / urine for ZIKV RNA 
detection, which will be performed simultaneously.  
b. 
Collect blood for TORCH testing and syphilis (RPR), if clinically indicated, and send to the local laboratory. (Do not repeat previously positive tests.) 
12. Record results from interim fetal USs performed according to country and local clinical guidelines since the prior study visit. 
13. Administer at continental U.S. sites only if this is an “unplanned symptomatic” visit. 
 
 
 

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Appendix II 
Infant Schedule of Evaluations  
Birth  
(+ 48 hours) 
3 Months  
(week 12 +/- 2 weeks) 
6 Months  
(week 24 +/- 2 weeks) 
12 Months  
(week 52 +/- 2 weeks) 
IC (Obtain from parent(s)/legal guardian(s) or 
confirm if previously obtained) 
X 
 
 
 
Health history (include results from laboratory 
tests conducted for clinical care, if applicable) 
X 
X 
X 
X 
PE 1 
X 
X 
X 
X 
Ophthalmologic exam 2 
X 
X 
X 
X 
Neurological exam 
X 
X 
X 
X 
Hearing assessment 3 
X 
X 
X 
X 
HIV-related test results and treatments, if 
applicable 4 
X 
X 
X 
X 
NEURODEVELOPMENTAL/NEUROPSYCHOLOGICAL EVALUATIONS 
ND assessment for screening options: HINE; 
ASQ; BSID-III 
 
X 
X 
X 
ND assessment for diagnostics: BSID-III 5 
 
X 
X 
X 
NEUROIMAGING RESULTS ABSTRACTION/ASSESSMENT 
Head US (abstract results) 6 
X 
X 7 
X 
X 
Head CT/MRI scan (abstract results) 6 
X 
X 
X 
X 
LABORATORY EVALUATIONS * 
(Blood volumes within parentheses are collected only if indicated.) 
Blood: 
        ZIKV RNA detection 
        ZIKV IgM Abs 
DENV IgM Abs (CKNV and WNV, as 
needed) 
4 ml 
3ml 
3ml 
3ml 
Blood: TORCH (blood or urine for CMV) & 
syphilis (RPR) (see the HIV ZIP MOP for 
asymptomatic newborn testing guidance) 8 
(3 ml) 
 
 
 
Urine: ZIKV RNA detection 
X 
X 
X 
X 
BIOREPOSITORY SPECIMENS (if consented for future use) 
Urine 
X 
X 
X 
X 
Remnant CSF 9  
X 
X 
X 
X 
TOTAL BLOOD VOLUMES  
(max. volume per visit = 3 ml per kg of weight) 
Min 4 ml 
Max 7 ml 
Min 3 ml 
Max 3 ml 
Min 3 ml 
Max 3 ml 
Min 3 ml 
Max 3 ml 

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Footnotes: 
* In case of low yield and/or low birthweight, prioritize blood specimen collection for ZIKV testing. Maximum of 3 ml per kg of weight (3.8% of total blood volume).  
 Lower limits for sick children are advisable. Refer to the HIV ZIP MOP for additional guidance on maximum blood volumes and laboratory testing algorithms. 
 
1.  Not required if a PE was performed within the past 4 weeks. Head circumference must be included. 
2.  If ophthalmology exam is abnormal, refer for additional testing and collect results from any tests done for clinical reasons. 
3.  If hearing assessment is abnormal, refer to audiology and collect results from any tests done for clinical reasons. 
4.  Abstract HIV diagnostic testing results, and if applicable, interim HIV VL and T-cells results. 
5.  Perform diagnostic ND assessments if the screening assessment is abnormal. 
6. Abstract results of neuroimaging assessments whenever they are performed. Does not need to coincide with the schedule of evaluations timeline. Do not perform for the 
study, except head US, as indicated below.  
7. ZIKV-exposed infants only: Perform a head US for the study only if none was performed by the age of 3 months.  
8. Only at birth if any individual tests are indicated. 
9. From specimen done for clinical care only.  
 
 

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Appendix III 
List of Participating Sites and Investigators 
Site Address 
Principal Investigator 
Telephone / Email 
Brazil  
Institute of Pediatrics Martagao 
Gesteira Federal University of Rio de 
Janeiro 
Rua Bruno Lobo, 50 
Ilha do Fundão 
Rio de Janeiro, 21941-912 
Brazil 
 
Site 5071 
 
Cristina Hofer, MD 
 
Email: cbhofer@hucff.ufrj.br 
Phone: 55-21-3148-2255 
 
 
 
 
 
Hospital Federal dos Servidores do 
Estado 
Centro de Pesquisa Clinica, 
DIP – Serviço de Doenças 
Infecciosas e Parasitarias, Rua 
Sacadura Cabral 
178, anexo IV, 4 andar, Saude 
Rio de Janeiro, 20221-903 
Brazil 
 
Site 5072 
 
Esaú Custódio João, MD, PhD 
 
 
 
Dra. Maria Isabel Gouvea, 
MD, PhD 
 
Email: esaujoao@gmail.com 
Phone: 55-21-2233-0018 
 
 
Email: : bebelsgouvea@uol.com.br 
Phone: 55-21-2233-0018 
 
Federal University of Minas Gerais 
School of Medicine 
Avenida Alfredo Balena 190 
Room 161 
Belo Horizonte, Minas Gerais, 
30130-100 
Brazil 
 
Site 5073 
 
Jorge Andrade Pinto, MD 
 
Email: jorgeandradepinto@gmail.com 
mailto:jpinto@medicina.ufmg.br 
Phone: 55-31-3409-9822 
Ribeirão Preto Medical School 
Department of Pediatrics 
University of Sao Paulo 
Avenida Bandeirantes 3900 
Ribeirão Preto 
Sao Paulo, 14049-900 
Brazil 
 
Site 5074 
 
Marisa M. Mussi-Pinhata, 
MD 
 
Email: mmmpinha@fmrp.usp.br 
Phone: 55-16-3602-2807 

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Site Address 
Principal Investigator 
Telephone / Email 
Hospital Geral De Nova Igaçu Brazil 
953 Avenida Henrique Duque 
Estrada Mayer  
Alto da Posse 
Nova Iguaçu, Rio de Janeiro, 26050-
210 
Brazil 
 
Site 5097 
 
Jose da Silva Pilotto, MD, 
PhD 
 
Email: pilotto@uninet.com.br  
          jhpilotto@gmail.com 
Phone: 55-21-98182-9797 
 
United States, including Puerto Rico  
San Juan Hospital Research Unit 
PMB #128, GPO Box 70344 
San Juan, Puerto Rico 00936 
 
Site 5031 
 
Midnela Acevedo, MT, MD 
 
 
 
Rodrigo Díaz-Velasco, MD, 
FACOG 
 
Email: 
macevedo@SanJuanciudadPatria.com 
Phone: 787-765-4186  
 
Email: rodrigodiazvelasco@gmail.com 
Phone: 787-765-4186 
Children’s Diagnostic & Treatment 
Center, Inc. 
1401 South Federal Highway 
Ft. Lauderdale, FL 33316 
 
Site 5055 
 
Ana M. Puga, MD 
 
Email: apuga@browardhealth.org 
Phone: 954-728-1050  
 
Bronx-Lebanon Hospital Center 
1685 Morris Avenue, Suite 1G 
Bronx, NY 10457 
 
Site 5114 
 
Murli U. Purswani, MD 
 
Email: mpurswan@bronxleb.org 
Phone: 718-960-1010  
 
University of Miami  
Pediatric Infectious Diseases and 
Immunology 
Batchelor’s Children’s Research 
Institute 
1580 NW 10th Avenue, Suite 286 
Miami, FL 33136 
 
Site 4201 
 
Gwendolyn Scott, MD 
 
 
 
 Charles Mitchell, MD 
 
Email: gscott@med.miami.edu 
Phone: 305-243-6522 
 
 
Email: cmitchel@med.miami.edu 
Phone: 305-243-2755 
 
University of Puerto Rico Pediatric 
HIV/AIDS Research Program 
Medical Sciences Campus 
Main Building, 3rd floor, Room 374 
School of Medicine  
San Juan, Puerto Rico 00935 
 
Site 6601 
 
Zoe Rodriguez, MD 
 
Email: zoe.rodriguez@upr.edu 
Phone: 787-645-0937   
 

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Appendix IV 
Sample Maternal Informed Consent 
See attached separate document 
 
 
Appendix V 
Sample Consent for Infant Participation 
See attached separate document