Long-Term Human Papillomavirus Vaccination Effectiveness and Immunity in Rwandan Women Living With and Without Human Immunodeficiency Virus
Conditions: Cervical Cancer, Human Papilloma Virus, Human Immunodeficiency Virus, HPV-Related Carcinoma
Sponsor: Montefiore Medical Center

Protocol: Long-term Human Papillomavirus Effectiveness and Immunity in Rwandan Women Living With 
and Without Human Immunodeficiency Virus
NCT05247853
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Long-Term Human 
Papillomavirus Vaccination 
Effectiveness and Immunity in 
Rwandan Women Living with 
and Without Human 
Immunodeficiency Virus
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INVESTIGATORS
Co-Principal Investigators:
Kathryn Anastos, MD
Professor of Medicine and Epidemiology & Population Health 
Albert Einstein College of Medicine and Montefiore Medical Center
Léon Mutesa, MD, PhD 
Professor/Director Center for Medical Genetics 
College of Medicine and Health Sciences 
University of Rwanda
Adebola Adedimeji, PhD, MBA
Associate Professor, Department of Epidemiology and Population Health
Albert Einstein College of Medicine and Montefiore Medical Center
Marcel Yotebieng, MD, PhD, MPH
Department of Medicine 
Albert Einstein College of Medicine and Montefiore Medical Center
Gad Murenzi, MD, MPH
Country Principal Investigator
Einstein-Rwanda Research & Capacity Building Program
Research for Development (RD Rwanda) and Rwanda Military Hospital
Key Personnel: 
Philip E. Castle, PhD, MPH
Division of Cancer Epidemiology & Genetics
National Cancer Institute (USA)
Gallican Kubwimana
Einstein-Rwanda Research & Capacity Building Program
Research for Development (RD Rwanda) and Rwanda Military Hospital
Fabienne Shumbusho, MD
Einstein-Rwanda Research & Capacity Building Program
Research for Development (RD Rwanda) and Rwanda Military Hospital
Athanase Munyaneza, RN, MPH
Einstein-Rwanda Research & Capacity Building Program
Research for Development (RD Rwanda) and Rwanda Military Hospital
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Julia C. Gage, PhD, MPH
Division of Cancer Epidemiology & Genetics
National Cancer Institute (USA)
Brad Aouizerat, PhD
Colleges of Nursing and Dentistry
New York University
Robert D. Burk, MD
Departments of Pediatrics, Microbiology & Immunology, Obstetrics & Gynecology and 
Women's Health and Epidemiology & Population Health
Albert Einstein College of Medicine and Montefiore Medical Center
Jose Jeronimo, MD
Division of Cancer Epidemiology & Genetics
National Cancer Institute (USA) 
Lynn Murchison, MPH
Department of General Internal Medicine
Albert Einstein College of Medicine and Montefiore Medical Center
Natasha Hansen, MPH
Division of Cancer Epidemiology & Genetics
National Cancer Institute (USA)
 
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ACRONYMS
Adenocarcinoma in situ (AIS)
Age Standardized Rates (ASR)
Albert Einstein College of Medicine (Einstein)
American Society for Colposcopy and Cervical Pathology (ASCCP)
Anal Intraepithelial Neoplasia (AIN)
AIN Grade 2 (AIN2)
AIN Grade 3 (AIN3)
AIN2 or More Severe Diagnoses (AIN2+)
AIN3 or More Severe Diagnoses (AIN3+)
Antiretroviral Therapy (ART)
Audio Computer-Assisted Self-Interview (ACASI) 
Central Africa International Epidemiology Databases to Evaluate AIDS (IeDEA)
Cervical Intraepithelial Neoplasia Grade 3 (CIN3)
Cervicovaginal microbiome (CVM)
CIN Grade 2 (CIN2)
CIN Grade 3 (CIN3)
Confidence interval (CI)
Formalin-fixed paraffin-embedded (FFPE)
Frederick National Laboratory of Cancer Research (FNLCR)
Generalized estimating equations (GEE)
Geometric mean titers (GMT)
High-resolution anoscopy (HRA)
High-risk HPV (hrHPV)
HIV-negative (HIV[-])
Human Immunodeficiency Virus (HIV)
Human Papillomavirus (HPV)
Infrared coagulation (IRC)
Invasive Cervical Cancer (ICC)
Loss to follow-up (LTFU)
Ministry of Health (MoH)
Next-generation Sequencing (NGS)
U.S. National Cancer Institute (NCI)
Participant Identification Number (PID)
People Living with HIV (PLWH)
REDCap (Research Electronic Data Capture)
Research for Development (RD Rwanda)
Rwanda Military Hospital (RMH)
Sub-Saharan Africa (SSA)
University of Rwanda (UR)
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U.S. Centers for Disease Control and Prevention (CDC)
Virus-like particles (VLPs)
WLWH (Women Living with HIV)
World Health Organization (WHO)
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Table of Contents
EXECUTIVE SUMMARY ......................................................................................................1
I.INTRODUCTION ................................................................................................................9
I.A. Epidemiology and Natural History of Human Papillomavirus (HPV) and Cervical Cancer .........................9
I.B. Prophylactic HPV Vaccination....................................................................................................................10
I.C. Prophylactic HPV Vaccination in People Living with HIV (PLWH)..........................................................11
I.D. HPV Vaccination Program in Rwanda ........................................................................................................12
II.RATIONALE ....................................................................................................................13
III. SPECIFIC AIMS............................................................................................................14
IV. SIGNIFICANCE.............................................................................................................14
V. RELATIONSHIPS, COLLABORATIONS AND ROLES..................................................15
VI. APPROACH....................................................................................................................16
VI.A. Study Design.............................................................................................................................................16
VI.B. Study Locations ........................................................................................................................................16
VI.C. Target Population......................................................................................................................................17
VI.D. Eligibility Criteria.....................................................................................................................................18
VI.D.1 Inclusion Criteria................................................................................................................................18
VI.D.2. Exclusion Criteria..............................................................................................................................18
VI.E. Recruitment and Eligibility Screener ........................................................................................................18
VI.F. Study Visits ...............................................................................................................................................19
VI.F.1. Enrollment Visit.................................................................................................................................19
VI.F.1.a. Informed Consent, HIV testing and Study Questionnaire ...............................................................19
VI.F.1.b. Blood Collection .............................................................................................................................21
VI.F.1.c. Cervicovaginal, Anal, and Oral Specimens.....................................................................................21
VI.F.1.d. Biospecimen Transport and Storage ...............................................................................................22
VI.F.2. Follow-Up Visit of the HPV-Positive Cohort ....................................................................................22
VI.G. Study Test Results and Clinical Management ..........................................................................................24
VI.G.1. Management of Persistent Cervicovaginal HPV ...............................................................................24
VI.G.2. Management of Persistent Anal HPV................................................................................................25
VI.G.3. Management of Persistent Oral HPV ................................................................................................26
VI.G.4. Study Exit..........................................................................................................................................26
VI.H. Biospecimen Processing, Storage, and Shipment .....................................................................................26
VI.I. Laboratory Testing.....................................................................................................................................27
VI.I.1. AmpFire HPV Testing........................................................................................................................27
VI.I.2. HPV Antibody Titers..........................................................................................................................27
VI.I.3. CVM Analyses....................................................................................................................................28
VI.I.4. Pathology ............................................................................................................................................28
VII. STATISTICAL SECTION .............................................................................................28
VII.A. Specific Aim 1.........................................................................................................................................28
VII.A.1. Analyses...........................................................................................................................................28
VII.A.2. Sample Size/Power for Gardasil®-vaccinated vs. unvaccinated WLWH........................................30
VII.B. Specific Aim 2.........................................................................................................................................31
VII.B.1. Analyses...........................................................................................................................................31
VII.B.2 Sample Size/Power to compare Gardasil®-vaccinated WLWH vs. Gardasil®-vaccinated HIV[-] 
women............................................................................................................................................................31
VII.C. Exploratory Aim: A Study of 6-12 Month Type-Specific HPV Persistence ...........................................31
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VIII. CAPACITY BUILDING AND TECHNOLOGY TRANSFER .......................................32
IX. RECRUITMENT AND RETENTION .............................................................................33
IX.A. Community Engagement ..........................................................................................................................33
IX.B. Benefits to Participation............................................................................................................................33
IX.C. Barriers to Participation ............................................................................................................................33
IX.D. Informational Materials ............................................................................................................................33
IX.E. Planning and Timeline ..............................................................................................................................34
IX.F. Recruitment Strategies ..............................................................................................................................34
IX.G. Retention Strategies..................................................................................................................................34
IX.H. Diversity ...................................................................................................................................................34
IX.I. Staff............................................................................................................................................................35
X.PROTECTION OF HUMAN SUBJECTS..........................................................................35
X.A. Risks to Human Subjects ...........................................................................................................................35
X.A.1. Human Subjects Involvement, Characteristics, and Design ...............................................................35
X.A.2. Study Procedures, Materials, and Potential Risks...............................................................................36
X.B. Adequacy of Protection Against Risks .........................................................................................................39
X.B.1. Informed Consent and Assent.............................................................................................................39
X.B.2. Protections Against Risk.....................................................................................................................39
X.C. Potential Benefits of the Proposed Research to Research Participants and Others ....................................41
XI. LIMITATION AND FUTURE DIRECTIONS ................................................................41
XII. DISSEMINATION OF RESEARCH RESULTS............................................................42
XIII. TIMELINE ..................................................................................................................42
APPENDIX I. VISUAL SUMMARY OF THE STUDY PROTOCOL.....................................43
APPENDIX II. WRITTEN INFORMED CONSENT DOCUMENT......................................44
APPENDIX III. ENROLLMENT QUESTIONNAIRE..........................................................53
APPENDIX IV. FOLLOW UP QUESTIONNAIRE ..............................................................59
REFERENCES.....................................................................................................................61
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EXECUTIVE SUMMARY
Cervical cancer is the 4th most common cancer and cause of cancer-related death in women 
globally; in many lower-resource settings, especially sub-Saharan Africa (SSA), it is the most 
common. Virtually all cervical cancer and precancer are caused by 12-15 high-risk human 
papillomavirus (HPV) types. HPV16 causes approximately 55-60% and HPV18 causes 
approximately 10-15% of cervical cancer while the remaining 12 HPV types cause the remaining 
25-30% of cervical cancer. High-risk HPV, predominately HPV16, also causes most anal, vulvar, 
vaginal, and penile cancers and a significant proportion of oropharyngeal cancers. Prophylactic 
HPV vaccines have been developed and have been shown to be nearly 100% protective against 
incident HPV infection and related abnormalities in the general population. However, the evidence 
for the effectiveness of prophylactic HPV vaccines in women living with human 
immunodeficiency virus (HIV) (WLWH) is less clear. HIV infection increases the risk of cervical 
cancer due to an impaired immune response to HPV. 
Rwanda is a high-burden cervical cancer country where the prevalence of HIV is 3.7% among 
adult women, with higher HIV prevalence among young women (1). In 2011, Rwanda 
implemented a national HPV vaccination program with Gardasil®, which protects against 
HPV16 and HPV18, the two HPV types that cause ~70% of cervical cancer, and HPV6 and 
HPV11, the two types that cause ~90% of anogenital warts (HPV6/11/16/18). Their program has 
achieved >90% coverage of the target population, primarily girls aged 12 years, annually. The 
implementation of a highly successful HPV vaccination program and the high prevalence of 
HIV, in addition to the research and medical capacity that Albert Einstein College of Medicine 
(Einstein) has helped to build at the Rwanda Military Hospital (RMH), Research for 
Development (RD Rwanda) and University of Rwanda (UR), makes Rwanda the ideal locale to 
study the long-term effects of HPV vaccination in WLWH.
To answer questions about HPV vaccine effectiveness and immunity in Rwandan WLWH, 
collaborators at Einstein, RD Rwanda, RMH, and UR will conduct an observational study of 
WLWH and HIV-negative (HIV[-]) women who did (birth cohorts 1996 and later) and WLWH 
who did not (birth cohorts before 1996) receive HPV vaccination through the national vaccination 
program. We will compare cervicovaginal, anal, and oral prevalent and 6-12 month persistent 
HPV6/11/16/18 infections in HPV-vaccinated and -unvaccinated WLWH and HPV-vaccinated and 
-unvaccinated HIV[-] women (n=757 per group; 4 groups; 3,028 women total). We will also 
compare the HPV immune response in at least 548 HPV-vaccinated WLWH to at least 548 HPV-
vaccinated HIV[-] women and the impact of switching from 3 doses to 2 doses of Gardasil® in 
2015. Finally, we will investigate the risk factors, including the cervicovaginal microbiome, for 
prevalent and 6-12 month persistent HPV infection in young WLWH and HIV[-] women. Our 
long-term goal is to establish a cohort of WLWH in whom we can examine the long-term 
effectiveness of HPV vaccination in WLWH now and in the future. This contribution is significant 
as it will establish the population effectiveness of HPV vaccination in WLWH living in SSA, the 
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group of women at the highest risk of cervical cancer, for which there is a dearth of evidence. The 
proposed research is innovative as it leverages and expands the local research and medical capacity 
in Rwanda to examine one of the critically unanswered questions about HPV vaccine effectiveness 
in the context of the World Health Organization’s (WHO) call for the elimination of cervical 
cancer.
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I.INTRODUCTION 
I.A. Epidemiology and Natural History of Human Papillomavirus (HPV) and Cervical 
Cancer
Cancer of the uterine cervix, invasive cervical cancer (ICC), with an estimated annual incidence 
of 570,000 cases and 311,000 related deaths, is the 4th most common female cancer and cause of 
cancer-related mortality in females globally (2). The introduction of effective Papanicolaou 
(Pap)-based (cytology) screening in some, mostly high-income, countries in the mid- to late-20th 
century has led to a steady decrease in cervical cancer incidence and mortality in those countries 
(3, 4). More than 80% of all cervical cancer occurs in low- and middle-income countries (2), 
where high-coverage, effective cytology-based screening has never been successfully 
implemented (3, 4).
Africa, specifically SSA, suffers the greatest burden of ICC. Approximately one-fourth of all 
ICC and ICC-related deaths globally occur in Africa. More than 90% of ICC in Africa occurs in 
SSA, which has an ICC incidence age-standardized rate (ASR) of 34.8 per 100,000 and mortality 
ASR of 24.1 per 100,000 (2). Rwanda has ICC burden similar to SSA with incidence ASR of 
31.9 per 100,000 and mortality ASR of 24.1 per 100,000 (4). In addition to a lack of capacity to 
implement Pap-based screening there, women living in SSA suffer from many other infectious 
disease epidemics, including human immunodeficiency virus (HIV). Cervical cancer was 
included as an acquired immunodeficiency syndrome (AIDS)-defining disease in adolescents and 
adults in 1993 (5-7).
WLWH have a significantly elevated risk of cervical cancer (8, 9), due to an impaired immune 
response to HPV, compared to HIV-uninfected (HIV[-]) women. People living with HIV 
(PLWH) are also at an increased risk of other HPV-related malignancies, notably anal and 
oropharyngeal cancers (8, 10). HIV infection is an important contributor to the unequal burden of 
cervical cancer in SSA due to its high prevalence compared to the rest of the world (11-13). 
Many SSA countries have a HIV prevalence among those aged 15-49 years of >5% and even 
>20% (14). In Rwanda, it is estimated that 3.0% (1) of the adult population are living with HIV 
with a prevalence of 1.8% among women aged 20-24 and 3.8% among women aged 25-29 years 
(15).
Persistent cervical infections by high-risk HPV types cause virtually all ICC and its immediate 
precursor lesions, e.g., cervical intraepithelial neoplasia grade 3 (CIN3) and adenocarcinoma in 
situ (AIS), everywhere in the world (16, 17). HPV causes most anal and vaginal cancer and a 
significant proportion of vulvar, penile, and oropharyngeal cancers (18). HPV16 is the most 
important, responsible for approximately 55-60% of ICC (19). HPV18 is the next most 
important, responsible for approximately 10-15% of ICC, including 30% of adenocarcinoma of 
the cervix (19), which is on the rise in Western Countries (20, 21). Together, HPV16 and HPV18 
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account for approximately 70% of ICC and the same ~15 high-risk HPV types account for ~99% 
of ICC everywhere in the world (19).
The natural history of HPV and cervical carcinogenesis can be represented by a simple, causal 
schema of four, reliably measured stages (22): 1) HPV acquisition, 2) HPV persistence, 3) 
progression to precancer (CIN3/AIS), and 4) ICC. The key step in cervical carcinogenesis is 
overt, measurable HPV persistence, which even after a year or two strongly predicts the 
development of cervical precancer and cancer (i.e., CIN3 or more severe diagnoses) (CIN3+) 
(23). HIV co-infection has a profound impact on the natural history of HPV, thereby increasing 
the risk of ICC. HIV co-infection increases the 1) likelihood of cervical and anal HPV 
persistence and 2) likelihood of cervical and anal HPV infections progressing to precancer (24, 
25). Incident HIV infection also significantly increases the risk of newly detected cervical and 
anal HPV infection, presumably due in part to immune dysregulation resulting in increased 
persistence of even transient infections (prevalence = incidence * duration) and/or and re-
activation of a latent/well-controlled HPV infection (25).
Meta-analyses of HIV/AIDS cohorts reported a 6-fold increased incidence of cervical cancer 
compared to the general female population/HIV[-] women (26). Unexpectedly, despite the 
introduction of combined anti-retroviral therapy (ART) and concomitant immune reconstitution, 
the incidence of the HPV-related malignancies, notably cervix and anal carcinomas, has not 
decreased as they have for other malignancies (e.g., Kaposi sarcoma and specific types non-
Hodgkin lymphoma i.e., Burkitt Lymphoma, Immunoblastic Lymphoma, and primary 
lymphoma of the brain) and may have increased. This increase is perhaps the result of HIV-
infected patients living longer (27, 28), long enough to develop HPV-related cancers (29, 30).
I.B. Prophylactic HPV Vaccination
The discovery of high-risk HPV (hrHPV) infections as the necessary cause of cervical cancer has 
led to revolutionary advances in cervical cancer prevention, including the development of 
prophylactic HPV vaccines for primary prevention. Current prophylactic HPV vaccines are 
based on the self-assembly of recombinantly expressed L1 protein in cell lines into virus-like 
particles (VLPs) that resemble native viral capsids but without the viral genome necessary for 
viral replication.
The first generation of HPV vaccines, Gardasil® (Merck & Co, Kenilworth, NJ, USA) (31) and 
Cervarix™ (GlaxoSmithKline, Wavre, Belgium) (31), targeted HPV16 and HPV18 (HPV16/18). 
Gardasil® also targets HPV6 and HPV11 (HPV6/11), non-high-risk HPV types responsible for 
90% of anogenital warts (Condyloma acuminata). The next generation HPV vaccine, Gardasil® 
9 (Merck & Co) (32), targets HPV31, 33, 45, 52, and 58 in addition to HPV6, 11, 16 and HPV18 
and is predicted to prevent ~90% of cervical cancers globally.
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Given that HPV vaccination is only prophylactic and not therapeutic, the ideal timing of HPV 
vaccination is prior to sexual initiation and exposure to HPV i.e., HPV-naïve women. On a 
population level, this can be achieved by vaccinating a few years before the population median 
age of sexual initiation. As the median age of sexual initiation is typically 15-17 years in many 
populations, the World Health Organization (WHO) recommends vaccination programs to target 
9-13-year-old girls (33). With older cohorts of women, prophylactic vaccines are equally 
efficacious but less effective i.e., fewer women benefit from HPV vaccination, resulting in lower 
effectiveness and cost-effectiveness, because some women have already been infected by HPV 
(34-36). Based on immunogenicity data, WHO (37) and the U.S. Centers for Disease Control and 
Prevention (CDC) (38, 39) now recommend two-dose schedules for those under the age of 15 
years and three doses for those 15 years and older for all HPV vaccines.
There are ample data demonstrating efficacy and population effectiveness of Gardasil® against 
HPV6, 11, 16, and 18 (HPV6/11/16/18) infections, anogenital warts (40), related high-grade 
cervical abnormalities (41), and now even cancer in immune-competent populations (41). Several 
countries, e.g. Australia (42-48), Scotland (49), Denmark (50), and the U.S. (51-53), were early 
adopters of HPV vaccination and have documented reductions in HPV infections and -related 
diseases and abnormalities. A meta-analysis on the impact of HPV vaccination found reductions 
in anogenital warts, HPV infections, CIN grade 2 (CIN2) or more severe diagnoses (CIN2+) 
among girls and women, and on anogenital warts diagnoses among girls, women, boys, and men 
(40). A report from Finland provided the first evidence that HPV vaccination prevents cervical 
cancer (41). Recent reports from Finland, Sweden, and Denmark now provide real-world 
evidence that HPV vaccination prevents cervical cancer (41, 54, 55). 
I.C. Prophylactic HPV Vaccination in People Living with HIV (PLWH)
Data on HPV vaccine efficacy and effectiveness data are lacking for WLWH (8, 9). To date, 
most studies of HPV vaccine in WLWH focused on immunogenicity and safety. Generally, HPV 
vaccination in PLWH has been well tolerated, safe, and resulted in good immune responses. A 
study of Gardasil® of children living with HIV and another study of WLWH reported high 
seroconversion to Gardasil (56, 57). The same study in children living with HIV showed high 4-
year persistent HPV type-specific immunity (58) and immune memory cells as well as a 
significant increase in, and persistence of, antibody titers following a 4th dose (59). 
Studies have noted an impact of HIV disease status (CD4 counts and viral suppression) on the 
immune responses to HPV vaccination. Studies have reported lower seroconversion and antibody 
titers in WLWH not taking (vs. taking) antiretroviral therapy (ART) and with lower (vs. higher) 
CD4 counts (57, 59). Similarly, another study found peak antibody titers to be 2- to 3-fold higher 
in mid-adult WLWH with full HIV viral suppression compared to those not suppressed (60). 
Interestingly, higher anti-HPV18 titers but similar anti-HPV16 titers were reported in response to 
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HPV vaccination by Cervarix® compared to Gardasil® in PLWH but the differences in the 
former was primarily due to differences in the immune responses in the WLWH (61).
The few studies of HPV vaccine effectiveness in WLWH provided promising but inconclusive 
data on immunogenicity. However, these completed studies of HPV vaccination in PLWH have 
not documented any measurable efficacy or effectiveness, and notably long-term effectiveness. 
Immunogenicity studies in WLWH have been of insufficient sample size to address 
efficacy/effectiveness. Studies of HPV vaccination in select PLWH at high risk of anal cancer 
have been limited in sample size because of the high HPV anal exposure (and likely 
misclassification of exposure) to targeted HPV types prior to enrollment (62) so that few truly 
incident events could be observed. Of those studies that did have endpoints, one recent trial of 
Gardasil® in PLWH aged 27 and older concluded that “This double-blind, randomized trial did 
not find a benefit from HPV vaccination to prevent anal HPV infection or anal high-grade 
squamous intraepithelial lesions” and favorable but inconclusive benefit for protection against 
oral HPV (63). Another study reported 5-fold higher incidence of abnormal cervical cytology 
among perinatally HIV-infected vs. HIV-exposed, uninfected youths (64).
ClinicalTrials.gov (65) reports several ongoing studies (i.e., enrollment listed as open) of 
assessing HPV vaccination in PLWH but none address prophylactic efficacy or long-term 
effectiveness. Several new projects are being launched as part of the “Prevention of HPV-related 
Cancers in HIV-infected Individuals: United States-Latin American-Caribbean Clinical Trials 
Network: Partnership Centers” (66) have HPV vaccination studies but are limited in scope and 
will not provide long-term effectiveness for HPV vaccination on cervicovaginal or anal HPV 
infection in WLWH. 
The paucity of HPV vaccine effectiveness data in WLWH is especially a concern for the 
prevention of cervical cancer in SSA where cervical cancer is the most common cause of cancer 
death in women. Almost one quarter of the global burden of cervical cancer occurs in SSA and 
an estimated 20% of cervical cancer in SSA is attributable to HIV coinfection (26). Evidence of 
the protective effects of HPV vaccination in SSA WLWH are needed, especially since most 
long-term public health planning to address cervical cancer in SSA depends on the unproven 
effectiveness of these vaccines.
I.D. HPV Vaccination Program in Rwanda
In 2011-13, Rwanda, through a donation from Merck, launched a national HPV vaccination 
program. In 2011, over 92,000 girls in primary school grade six (~12 years old) were vaccinated 
with three doses of Gardasil®. During 2012 and 2013, a catch-up vaccination program targeted 
girls in secondary school grade three (~15 years old) (67). In 2014, HPV vaccination was 
supported by GAVI (68) and reverted to vaccinating 12-year-old girls (69). In 2015, Rwanda 
switched from 3 doses to 2 doses, 6 months apart, for vaccinating 12-year-old girls. In all years, 
Rwanda achieved ≥90% annual coverage with the recommended number of HPV vaccine doses 
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in the target population (67, 68, 70, 71). Thus, Rwanda is one of the earliest and most successful 
adopters of HPV vaccination globally. A summary of the HPV-vaccinated population is shown 
in Table 1.
Table 1. HPV vaccination data for Rwanda 
Year
N (HPV 
Vaccinated)
Approximate age 
at vaccination 
(years)
Birth year of 
vaccinated
Approximate 
age in 
2021/2023 
(years)
N HPV-
Vaccinated 
WLWH 
(estimated)
2011*
88,927
11-13
1998-2000
21-23/23-25
1,600
2012*
134,745
11-16
1996-2001
20-25/22-27
2,430
2013*
134,775
11-16
1997-2002
19-24/21-26
2,430
2014*
142,192
12
2002
19/21
2,560
2015**
106,522
12
2003
18/20
1,920
2016**
120,953
12
2004
17/19
2,180
2017**
120,594
12
2005
16/18
2,170
Total
848,708
15,290
*three doses of Gardasil®; **two doses of Gardasil®
Data provided by the Rwanda Ministry of Health
II.RATIONALE
In 2018, WHO called for the elimination of cervical cancer as a public health problem (72). To 
accomplish this goal, WHO put forth an ambitious strategic plan (73) that includes 90% of girls 
fully vaccinated against HPV by 15 years of age. Yet, we do not know if or how well HPV 
vaccination works in PLWH. Recent national and international guidelines have highlighted the 
lack of direct evidence of HPV vaccination in PLWH. Studies have demonstrated that HPV 
vaccines are safe and immunogenic in various groups of PLWH. Some studies demonstrated 
antibody levels lower in individuals with HIV compared to those without HIV, but the clinical 
significance of this observation is unknown (60, 74, 75).
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Given the high prevalence of HIV in SSA, it will be important to document both long-term 
effectiveness and immunity. Such data will inform strategies to optimize the impact of HPV 
vaccination in PLWH.
Rwanda is the ideal locale to answer questions about the long-term effectiveness and 
immunogenicity of Gardasil® in WLWH. The country has experienced nearly complete national 
coverage with Gardasil® since more than 10 years ago, starting in 2011, so predominately HPV-
vaccinated and unvaccinated WLWH are easily recruited based on birth year. Rwanda has 
excellent national vaccination records so that retrospectively the small number of participants 
misclassified by HPV vaccination status based on birth year subsequently can be correctly 
categorized. The prevalence of HIV in Rwanda is 2.7% among 15-30 year olds (1). The excellent 
HIV care program allows for easy and efficient recruitment and following of WLWH in an 
observational study of HPV vaccination impact. Rwanda is part of the Central Africa 
International Epidemiology Databases to Evaluate AIDS (IeDEA) consortium whose 
infrastructure can be leveraged to recruit and retain WLWH women in the proposed 
observational study. The Rwanda / US collaborative research group has a well-established, long-
term, and highly productive clinical and research presence in Rwanda, representing a unique 
opportunity to establish a prospective cohort study of the population effectiveness of HPV 
vaccination in WLWH (Section V).
III. SPECIFIC AIMS
We propose the following Specific Aims for our study of HPV vaccination in Rwandan 
WLWH: 
1. To measure population effectiveness of prophylactic HPV vaccine in reducing 
cervicovaginal, anal, and/or oral prevalent and 6-12 month persistent infections by 
HPV6/11/16/18; and 
2. To quantify, and examine the determinants of, long-term antibody (into young adulthood) 
responses to HPV vaccination
Exploratory Aim: Conduct a natural history study to investigate determinants, including 
cervicovaginal microbiome (CVM), of short-term HPV persistence in young WLWH and HIV[-] 
women living in a SSA setting.
IV. SIGNIFICANCE
The proposed study will provide the much-needed evidence regarding the long-term protection 
afforded by HPV vaccination in WLWH living in SSA, who are at the greatest risk of HPV-
related cancers. To our knowledge, there are no studies or trials to measure the effectiveness of 
HPV vaccination in WLWH living in SSA who were vaccinated at the optimal ages, prior to 
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sexual initiation. Most trials have focused on late teens and early adult PLWH, many of whom 
have already been exposed to HPV, and those trials have been conducted in high-income 
countries so the generalizability to low- and middle-income countries, and specifically SSA, is 
unclear. IARC/WHO is conducting a study of Gardasil® impact in Rwanda and Bhutan but that 
study is not specifically targeting WLWH, and therefore will have very few WLWH (67, 75, 76). 
Rwanda is one of the few places in the world, and the only place in SSA and low- and middle-
income countries, that the long-term effectiveness of HPV vaccination in WLWH can be 
examined.
Most studies of HPV vaccination impact focus on a single anatomic site and do not measure the 
multi-site exposure that is related to the total risk of HPV-related cancers. There is good but 
imperfect correlation of HPV infection at each anatomic site (77-80). Notably, the prevalence of 
anal HPV infection can be high in WLWH relative to HIV[-] women who are negative for that 
type in the cervix (77). Finally, this study will establish risk factors for early HPV type-specific 
persistence, a key step in the development of cervical precancer and cancer (16, 81, 82) in 
WLWH living in SSA.
V. RELATIONSHIPS, COLLABORATIONS AND ROLES
This study is continuing and leveraging the Einstein-Rwanda consortium and local capacity 
developed in a previous SSA Collaborative HIV and Cancer Consortia (U54) grant. The 
consortium has established capacity to conduct large epidemiological and clinical studies on 
HPV and anogenital cancer, colposcopy, and treatment of high-grade cervical abnormalities, 
high-resolution anoscopy (HRA) and treatment of high-grade anal abnormalities, and laboratory 
assays including the AmpFire assay and DNA extraction, all of which will figure prominently in 
the proposed study. Drs. Gad Murenzi (Co-I; Research for Developmet (RD Rwanda) & Rwanda 
Military Hospital [RMH]) and Philip Castle (Co-I; National Cancer Institute [NCI]) again will 
co-lead the cervical study as they did for the successful cervical project in the previous U54-
funded Einstein-Rwanda consortium. Dr. Anastos (PD/MPI; Einstein), PI of the Women’s 
Interagency HIV Study and the Central Africa International Epidemiology Databases to Evaluate 
AIDS, will provide leadership and expertise on conducting clinical and epidemiologic studies. 
Drs. Leon Mutesa (MPI; University of Rwanda [UR]), Brad Aouizerat (co-I; New York 
University), and Robert Burk (co-I; Einstein) will develop the local capacity for studies of the 
human microbiome. Drs. Anastos and Burk have collaborated for over 20 years in the Women’s 
Interagency HIV Study, with 40 shared publications. Drs. Castle and Anastos have worked 
together for 15 years to conduct studies of HPV in Rwandan women (83-86). Drs. Castle and 
Burk have collaborated for 20 years on molecular epidemiology studies of HPV, resulting in 
almost 50 publications together. Drs. Anastos and Aouizerat have collaborated for nearly 15 
years in studies WLWH in the United States.
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VI. APPROACH
VI.A. Study Design 
This prospective observational HPV natural history study will assess the long-term effects of 
Gardasil® on HPV6/11/16/18 infection among WLWH. At local health clinics in Kigali, 3,028 
women aged 18-28 years will be enrolled: 757 WLWH and 757 HIV[-] women who did receive 
HPV vaccination (birth cohorts 1996 and later) and 757 WLWH and 757 HIV[-] women who did 
not receive HPV vaccination (birth cohorts before 1996). The visual summary of the study is 
provided in Appendix I. At the enrollment visit, cervicovaginal, anal, and oral specimens will be 
collected and tested for HPV6/11/16/18 plus high risk 
HPV31/33/35/39/45/51/52/53/56/58/59/66/68 for exploratory aims. Participants positive for 
high-risk HPV and/or HPV6/11 on any sample will have a 6-12 month follow-up visit to 
measure HPV type-specific persistence - a surrogate endpoint recommended by the WHO (87). 
At young ages, the risk of precancer is very low, but for safety purposes, participants will be 
offered colposcopy if they have a persistent high-risk HPV cervical infection and anoscopy if 
they have a persistent HPV16/18 anal infection. Main outcomes will be prevalence and 6-12 
month type-specific persistence of cervicovaginal (as an excellent proxy for cervical sampling 
(87)), anal, and/or oral infections by HPV6/11/16/18 as well as antibody titers for 
HPV6/11/16/18. Cervical and anal biopsy specimens diagnosed as CIN2+ or AIN2+, 
respectively, will also be tested for the 15 high-risk and 2 low-risk HPV types for secondary 
outcomes. In an exploratory aim, we will examine risk factors, including the cervicovaginal 
microbiome (88), for HPV type-specific persistence in WLWH and HIV[-] women. 
At enrollment, blood will be collected for plasma from all participants. In the study laboratory, a 
stratified random sample of specimens will be selected to measure by ELISA anti-HPV16 and -
HPV18 antibody titers and compare HPV-vaccinated WLWH. We will test titers in at least 1,302 
samples (n≥548, 50% with three doses and 50% with two doses of Gardasil®, for both HPV-
vaccinated WLWH and for HPV-vaccinated HIV[-] women; n≥108 for both HPV-vaccinated 
WLWH and for HPV-vaccinated HIV[-] women as controls) and up to 3,028 samples.
VI.B. Study Locations
In Kigali, the study staff will work with five public clinics that participate in the International 
Epidemiology Databases to Evaluate AIDS (IeDEA) program and the WE-ACTx private clinic 
(Table 2). Study investigators and staff successfully worked with these clinics in a cervical 
cancer screening study of 5,000 WLWH (83). If needed, the staff will work with up to four other 
public health clinics to recruit additional participants.
Table 2. Study enrollment sites
IeDEA Sites
Non-IeDEA sites
1. Busanza health center (HC)
1. Cor-unum HC 
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2. Gikondo HC
3. Kicukiro HC
4. Nyarugunga HC
5. Rwanda Military Hospital
6. WE-ACTx private clinic
2. Kacyiru HC
3. Remera HC
4. Rwampara HC
VI.C. Target Population
This study will recruit women attending collaborating clinics and women from the communities 
surrounding those clinics. Three groups will be recruited:
●
757 HPV-vaccinated WLWH (Group 1), 
●
757 HPV-unvaccinated WLWH (Group 2), and 
●
757 HIV[-] women who are HPV-vaccinated (Group 3)
●
757 HIV[-] women who are HPV-unvaccinated (Group 4)
Within the HPV-vaccinated groups 1 and 3, the study will recruit at least 274 women with three 
doses and 274 women with two doses, with the remaining 209 will not be selected by the number 
of doses and may have 2 or 3 doses. The number of doses of HPV vaccination a woman received 
is dictated by introduction of HPV vaccination in Rwanda as previously shown in Table 1. In 
general, most Rwandan women born in 2003 or later will have been vaccinated with two doses 
of Gardasil®, women born between 1996 and 2002 will have been vaccinated with three doses of 
Gardasil®, and women born in 1995 or earlier will be unvaccinated.
Table 3. Target population by HIV status and age
Age at year of study 
enrollment (years) 
HIV Status
Approximate number 
of HPV vaccine doses
Birth year
in 2021
in 2023
N
3 doses 
1996-2002
19-25
21-26
274 
2 doses 
2003-2005
16-18*
18-20
274
2 or 3 doses
1996-2005
19-25
18-26
209
WLWH
0 doses - unvaccinated
1993-1995
26-30*
28-31*
757
3 doses 
1996-2002
19-25
21-26
274
2 doses 
2003-2005
16-18*
18-20
274
HIV[-]
2 or 3 doses
0 doses- unvaccinated
1996-2005
1993-1995
19-25
26-30*
18-26
28-31*
209
757
TOTAL
3,028
*Only women age 18-28 are eligible for enrollment. 
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Therefore, we will recruit by age, which will serve as a useful and very good proxy for HPV 
vaccination status, which otherwise would be difficult to confirm in real time at enrollment. HPV 
vaccination status then will be confirmed by retrospective review of the HPV vaccination records 
kept by the Rwanda Ministry of Health (MoH), who are collaborating on this project. The number 
of women recruited will therefore be stratified by age (as proxy for vaccine status) and HIV status 
(Table 3). 
VI.D. Eligibility Criteria
VI.D.1 Inclusion Criteria
1. Gender: Female
2. Age: 18-28 years. The age of inclusion criteria will likely be restricted as age-specific 
enrollment goals are met.
3. Physically and mentally able and willing to participate in the study.
4. Willing to provide written and signed or thumb printed, informed consent. 
5. Known to be living with HIV (i.e., enrolled in a treatment program), or consent to HIV 
testing to confirm HIV status. 
VI.D.2. Exclusion Criteria
1. Have positive pregnancy test or report to be pregnant at the time of visit or less than 6 
weeks post-partum (will be asked to make an appointment 6 or more weeks post-partum)
2. Report to be menstruating at the time of visit (will be asked to make new appointment)
3. History of hysterectomy and no longer have a cervix
4. History of treatment for cervical abnormalities after cervical screening 
5. History of cervical cancer
6. Report no previous sexual activity
7.
Because this study has age-specific enrollment goals for WLWH and HIV[-] women, once 
those enrollment goals are met for each study group, the respective cohorts will be closed 
and other eligible women will be excluded.
VI.E. Recruitment and Eligibility Screener
During the period of recruitment at a health clinic, WLWH and HIV[-] women will be solicited 
using advertisements throughout the entire clinic as well as outreach using community health 
workers (Figure 1). In addition, WLWH who are attending routine appointments at the HIV 
clinics will learn about the study during the group study overview sessions held in the health 
education room. All women who are interested in the study will meet with a study nurse who 
will give them an informational flyer and provide a brief verbal description of the study. The 
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study nurse will help the woman complete the eligibility screener that includes criteria listed 
above. If eligible and interested, the woman will register for an enrollment visit. 
Figure 1. Participant recruitment 
VI.F. Study Visits 
VI.F.1. Enrollment Visit 
VI.F.1.a. Informed Consent, HIV testing and Study Questionnaire
As outlined in Figure 2, at the enrollment visit conducted in a private exam room, the study 
nurse will describe the details of the study verbally and women will be provided an opportunity 
to ask any questions. The participant eligibility will be verified using the eligibility form. 
Women who report menstruating at the time of visit will be asked to make a new appointment 2 
weeks later. Women who report to be pregnant or less than 6 weeks post-partum will be asked to 
make an appointment 6 or more weeks post-partum. 
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Figure 2. Enrollment visit procedures 
Women will individually undergo the consenting process as described in Section X.B.1. Once 
the Informed Consent Form (Appendix II) is completed, a unique study participant 
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identification number (PID) will be assigned to the participant that will be used to identify all 
forms and specimens collected at the enrollment and follow-up visits.
Participants’ personal contact information will be confirmed. Participants will provide a urine 
sample for pregnancy testing to confirm they are not pregnant. Counseling will be provided pre 
and post testing as needed. Participants who are not known to be living with HIV will have a 
rapid HIV test with pre- and post-HIV-testing counseling following the National Rapid HIV 
Diagnostic Testing Protocol. Any participant who tests positive will be referred to the health 
center’s HIV clinic for HIV management following the Ministry of Health’s HIV management 
guidelines. The consent form obtains the participants’ permission to extract from medical records 
information related to their HIV care including CD4 count, viral load, ART use and age of 
initiation of HIV care. They will then complete a short questionnaire on socio-demographic 
information and risk factors for HPV including sexual behaviors (Appendix III). The 
questionnaire will be conducted in private, using an automated audio interview assistance tool 
Audio Computer-Assisted Self-Interview (ACASI) software. The study nurse will provide 
additional assistance only if requested.
VI.F.1.b. Blood Collection
For the study of anti-HPV16 and HPV18 antibody titers between WLWH and HIV[-] women 
(Specific Aim 2 listed above), the study nurse will collect one 5 mL tube of blood from all 
participants for HPV antibody serology testing. 
VI.F.1.c. Cervicovaginal, Anal, and Oral Specimens
For the oral specimen, the nurse will hand a medicine cup with the saline rinse to the participant. 
The nurse will start the timer and instruct the participant to gargle, swish, and spit. For 30 
seconds total, the participant will alternate between a 5-second swish and a 5-second gargle. The 
study nurse will help the subject count out the alternating 5 second squish and 5 second gargles 
at least 3 times. At the end of the 30 seconds, the study nurse opens the 5 oz. sterile specimen 
container and hands it to the participant, holding the top lid down to avoid contaminating it. The 
participant will spit the saline into the specimen container when done gargling. The study nurse 
will seal and label the specimen container with the participant ID.
Vaginal specimens will be collected using the AmpFire specimen collection brush (Atila 
BioSystems, Mountain View, CA, USA). The participant will lie on the exam table. The study 
nurse will insert the brush into the posterior vagina, turning the brush 3 turns left and right, 
before removing the brush. The study nurse will insert the brush into the collection tube and snap 
off the handle to break it. The study nurse will seal the tube and label it with the corresponding 
participant label.      
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Anal canal specimens will be collected using a scored Dacron swab. While the participant is 
lying on the exam table, the study nurse will collect the specimen by inserting a water-
moistened, scored Dacron swab into the anal canal, turning the swab 2-3 turns left and right, 
before removing the swab. The study nurse will insert the swab into the collection tube and snap 
off the handle at the score on the handle to break it. The study nurse will seal the tube and label it 
with the corresponding participant label.
VI.F.1.d. Biospecimen Transport and Storage
A study staff will transport the oral saline rinse, dry swabs, and blood samples to the study lab. 
Blood samples will be processed the same day they arrive at the lab. If same-day processing is 
not possible, whole blood will be stored at 4–8°C for up to 24 hours before the serum/plasma is 
separated. Aliquots of plasma will be stored at -80°C freezer for further testing. Serum will be 
discarded.
Oral specimens will be processed and tested for HPV genotypes. Upon arrival, samples will be 
stored at 4°C until processed. Aliquots will be stored frozen at -20°C for further testing. After 
testing, residual specimens will be discarded.
Dry anal and cervicovaginal specimens will be processed and tested using the Atila Ampfire 
HPV genotyping. Upon arrival, the oral rinse specimen containers will be stored at 4°C 
refrigerators for processing the next working lab day. Dry brushes and associated labels will be 
stored in a box in the freezer at -20ºC for processing the next working lab day. After testing, 
residual specimens will be aliquoted and stored frozen at -20°C for further testing. 
HPV genotyping will be conducted within 2 months of collection to allow ample time to 
schedule the 6-12 month follow up visit for women who test HPV positive for any HPV type at 
any tissue site.
VI.F.2. Follow-Up Visit of the HPV-Positive Cohort
Participants who test positive for any of the 17 HPV types on their cervicovaginal, oral, and/or 
anal specimen collected at baseline will be scheduled for a 6-12 month follow-up visit with a 
window of 5-15 months after their baseline visit; participants who test negative on all three 
specimens will exit the study. Objectives of the follow-up visit are to 1) measure 6-12 month 
HPV persistence, an accepted primary endpoint in prophylactic HPV vaccine trials (89), and 2) 
ensure the safety of the participants. As outlined in Figure 3, at the follow-up visit, the 
participant’s eligibility status will be confirmed. Participants will provide a urine sample for 
pregnancy testing to confirm they are not pregnant. Counseling will be provided pre and post 
testing as needed. Participants unknown to be living with HIV will have a rapid HIV test with 
pre- and post-HIV-testing counseling following the National Rapid HIV Diagnostic Testing 
Protocol. Any participant who tests positive will be referred to the health center’s HIV clinic for 
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HIV management following the Ministry of Health’s HIV management guidelines. Only 
specimens from those anatomic sites that were HPV positive at baseline will be collected 
utilizing the same protocol as at enrollment. A study staff will transport the oral saline rinse and 
dry swabs to the study lab to be stored until study testing for 17 HPV genotypes; the follow-up 
cervicovaginal and anal specimens will be tested within one month to ensure timely follow-up of 
high-risk individuals who need diagnostic services (colposcopy and/or anoscopy).
Figure 3. Activities at follow-up visit 
Follow-up Visit
Study staff confirms follow-up appointment and retrieves
participant folder
For samples HPV+ at enrollment, participant provides
1. Oral rinse specimen collection
2. Nurse-collected cervicovaginal specimen collection
3. Nurse-collected anal specimen collection
Clinic
Exam
Room
Does not
participate
that day
Eligibility form
is completed
Not eligible
Eligible
Receives pregnancy
counseling and
follow-up visit
rescheduled for
post-partum
Urine
pregnancy test
Pregnancy
test positive
Private
Room
•
Rapid HIV test with pre- and post-
counseling (participants not known HIV+)
•
Study questionnaire completed
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VI.G. Study Test Results and Clinical Management
VI.G.1. Management of Persistent Cervicovaginal HPV
A summary of the management of HPV-positive results is shown in Figure 4. To ensure the 
safety of participants, those who at the follow up visit, have a type-specific persistent HPV 
infection for one or more of the 15 hrHPV types (HPV16/18/31/33/35/39/45/51/52/53/56/58/ 
59/66/68), regardless of HIV status, will be referred to colposcopy. Colposcopy and treatment 
will be performed at RMH by a trained study physician or gynecologist on staff at the hospital.  
Digital images of the cervix will be taken using a study-provided contemporary digital camera 
(e.g., Samsung A21S or similar cellular phone camera). The participant’s images will be shown 
to her if she so desires. All images from a participant will be identified using a study ID. Images 
will not be analyzed at the clinic visit but be stored in the study secure NIH server immediately 
or, if not possible at the time, images will be uploaded when secure access to the study server 
becomes available. Images will be used for later analyses with automated visual evaluation 
algorithms being developed at the NCI for management of HPV-positive women (90). 
Biopsies will be taken of all acetowhite, visible cervical abnormalities consistent with the 
American Society for Colposcopy and Cervical Pathology (ASCCP) management guidelines 
(91). The colposcopy impression and location of biopsies will be recorded for clinical 
management files and saved in the participant record for study purposes. 
 
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Figure 4. Follow-up and Clinical Management of Baseline HPV-Positive Results
After undergoing colposcopy and biopsies, WLWH with persistent HPV16- or HPV18 infection 
will receive ablative treatment unless ineligible for ablative treatment per WHO Guidelines (92); 
those ineligible for ablation will receive treatment by LEEP. Biopsies will be read by two local 
pathologists and Dr. Hébert (Einstein). All participants diagnosed with CIN3+ will undergo 
treatment. In accordance with ASCCP guidelines, those diagnosed with CIN2 will not undergo 
immediate treatment due to the low risk of ICC following a CIN2 diagnosis, especially in young 
women, and the possible increased risk in negative reproductive outcomes such as pre-term 
delivery following treatment (10). They will be advised to seek clinical follow-up in a year.
After processing and reading according to local practices, formalin-fixed paraffin-embedded 
(FFPE) tissue specimens from biopsies and excisional treatment with results of CIN2+ will be 
sent to the study lab for HPV typing for study purposes (Section VI.I.1.).
To summarize, within the context of the research protocol, participants at risk of precancer will 
be provided with colposcopy as well as treatment and follow-up. Participants with a diagnosis 
that requires treatment beyond ablation or excision (i.e., invasive cervical cancer), will be 
referred and guided to appropriate local care. 
VI.G.2. Management of Persistent Anal HPV
WLWH with persistent HPV16 or HPV18 infection will undergo anoscopy; HIV[-] participants 
with persistent HPV16- or HPV18 infection will not undergo anoscopy due to their still low 
absolute risk of anal cancer (10) and the morbidity of treating anal abnormalities. Anoscopy and 
treatment will be performed at RMH by a study physician on staff at the hospital with training 
for this protocol.
A biopsy will be taken from acetowhite lesions. The anoscopy impression and location of 
biopsies will be recorded for clinical management files and saved in the participant record for 
study purposes. After processing and reading according to local practices, formalin fixed, 
paraffin embedded (FFPE) tissue specimens from biopsies and excisional treatment with results 
of anal intraepithelial neoplasia (AIN) grade 2 (AIN2) or more severe diagnoses (AIN2+) will be 
sent to the study lab for HPV typing analysis (Section VI.I.1.).
Those with a biopsy diagnosis of AIN grade 3 (AIN3) or anal cancer will be treated or referred 
for anal cancer management, respectively. Those diagnosed with AIN2 will not undergo 
immediate treatment due to the low risk of anal cancer following a AIN2 diagnosis and the 
aforementioned morbidity associated with treatment. Those with untreated AIN2 or persistent 
anal hrHPV by non-HPV16/18 types will be advised to seek clinical follow-up in a year. 
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Similar to colposcopy, within the context of the research protocol, participants at high risk of 
anal precancer will be provided with anoscopy as well as treatment and follow-up. Participants 
with a diagnosis that requires treatment beyond routine care (i.e., invasive anal cancer) will be 
referred and guided to appropriate local care. 
VI.G.3. Management of Persistent Oral HPV
There are no management guidelines or evidence-based intervention for type-specific 
persistent HPV. Therefore, participants will not receive any clinical intervention for type-specific 
persistent oral HPV.
VI.G.4. Study Exit
Other participants, including WLWH and/or those with persistent low-risk HPV6- or HPV11 
infection, will exit the study.
VI.H. Biospecimen Processing, Storage, and Shipment
The study lab will process, store and test for HPV in oral specimens in saline solution, dry 
samples, blood samples and FFPE tissue specimens. All specimens will be tested for individual 
17 HPV types using the AmpFire HPV genotyping test. 
In preparation for testing, oral specimens will be aliquoted, the dry sample will be rinsed into a 
vial with lysis buffer and FFPE tissue specimens will be suspended in lysis buffer. Specimens 
will be processed according to manufacturer’s instructions. All residual lysed specimens will be 
stored at the study lab at -80oC for later testing. Select samples will be sent to study 
collaborators’ laboratory for microbiome testing. Additional microbiome testing will be 
conducted at the study lab. 
Whole blood specimens will be processed to obtain aliquots of plasma. Following the protocol 
established by the Frederick National Laboratory of Cancer Research (FNLCR) HPV Serology 
Laboratory, select plasma specimens will be sent to FNLCR for testing and others will be tested 
at the study lab (Section V.I.2). After ELISA testing, residual plasma will also be stored at the 
study lab for later testing. 
A biospecimen bank containing residual material from samples collected will be established at 
the study lab for subsequent methodologic and etiologic research.
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VI.I. Laboratory Testing 
VI.I.1. AmpFire HPV Testing 
For HPV genotyping of cervicovaginal, anal, and oral specimens for Specific Aim 1 (Section 
VII.A), we will use the AmpFire HPV genotyping test (Genotyping High Risk HPV Real Time 
Fluorescent Detection; Atila Biosystems, Mountain View, CA, USA), which uses real-time PCR 
to detect 15 individual HPV genotypes HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66 
and 68, plus low-risk HPV6 and 11, in 5 assay reactions previously established at the study lab 
and validated against other commercially available assays at RMH (93-96). This method requires 
no transport buffer (dry specimens) and no DNA purification step and is well suited to be run in 
clinic settings by personnel with relatively minimal training. For oral specimens in saline 
solution, specimens will first be spun in the centrifuge to enrich the sample. The AmpFire system 
provides results for each type within 1.5 hours. Atila has agreed to provide a separate detection 
tube for the combined detection of HPV6 and 11 (HPV6/11) for this study. The assay will be run 
per the manufacturer’s instructions. The residual lysed specimen will be neutralized according to 
the manufacturer’s protocol using its buffer and stored at -20oC for long term storage and later 
testing.
FFPE tissues diagnosed as CIN2+ or AIN2+ will similarly have HPV genotyping using the 
AmpFire system according to the manufacturer’s protocol.
For monitoring of quality of Ampfire testing, aliquots of selected samples  will be sent to an 
independent lab for testing with a validated HPV typing assay. The results will be compared with 
local tests results for quality control and not clinical management. In some cases of negative Ampfire 
and the positive QC test, participants be referred for follow-up visit. Treatment will not be 
determined by the QC result. 
VI.I.2. HPV Antibody Titers 
For Specific Aim 2 (Section VII.B) anti-HPV16 and HPV18 IgG antibody titers will be 
measured from plasma by an ELISA based on a previously described method (97, 98) and 
according to the protocol established by the FNLCR HPV Serology Laboratory (99). Only anti-
HPV16 and HPV18 IgG antibody titers, and not titers for all 4 HPV types, will be evaluated as 
antibody titers are correlated between antibody responses for the different types. HPV16 and 
HPV18 are the two most important HPV types and cause approximately 70% of cervical cancers 
(19). HPV16 tends to induce the highest titers while HPV18 tends to induce the lowest titers 
among the 4 types (100, 101) and therefore measuring the two types gives us a range in 
responses while using study resources cost effectively. The FNLCR Serology Laboratory will 
teach the ELISA protocol to Dr. Johanna Daily (co-I; Einstein), who will oversee the transfer of 
this ELISA assay to the study lab in Rwanda.
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First, 20% of each group will be run at the Frederick NCI lab, then the results will be replicated 
at the study lab (masked to the original results), and then the remaining 80% of the testing will 
be finished at the study lab. As an additional quality control measure, a 10% random sample of 
specimens will be re-run at the NCI Frederick lab (masked to the original results) (inter-
laboratory reliability) and another 10% random sample of specimens at the study lab (intra-
laboratory reliability). The study lab will achieve at least 90% correlation for both anti-HPV16 
and -HPV18 titers with FNLCR Serology Laboratory on the first 20% of specimens tested before 
the study lab embarks on testing the last 80% of specimens (101). Positive (e.g., IS standards 
(102, 103)) and negative controls (e.g., negative plasma) will be included in some testing plates 
to monitor assay performance per WHO recommendations (104).
VI.I.3. CVM Analyses 
Baseline and follow-up specimens collected for baseline HPV-positive participants will be tested 
for cervicovaginal microbiota by next-generation sequencing (NGS) for our Exploratory Aim 
(Section VII.C.) on short-term HPV persistence. The first 20% of specimens will be tested in 
both the collaborating lab and the study lab until acceptable agreement is achieved (see below) 
after which the remaining 80% of specimens will be tested at the study lab with retesting 10% 
for quality control. 
VI.I.4. Pathology 
All histopathology slides will be scanned at the study lab and reviewed by two local pathologists 
and the Einstein study pathologist (105). If the diagnoses are concordant, no further review of the 
case will be performed. If the diagnosis is discordant and at least one pathologist diagnoses 
CIN2+, the biopsy slide will be subjected to a joint review and consensus diagnosis. For 
Negative/cervical intraepithelial neoplasia grade 1 pairs of diagnoses, which does not influence 
our analyses or the care of the participants, there will be no joint review. 
VII. STATISTICAL SECTION
VII.A. Specific Aim 1
 To measure population effectiveness of prophylactic HPV vaccine in reducing cervicovaginal, 
anal, and/or oral prevalent and persistent infections by HPV6/11/16/18.
VII.A.1. Analyses. 
We will calculate the point prevalence and the prevalence of persisting infection by individual 
HPV-type specific infections, with binomial 95% confidence intervals (95% CI), for each 
anatomic and for the woman (all anatomic sites) for Gardasil®-vaccinated WLWH, Gardasil®-
vaccinated HIV[-] women, unvaccinated WLWH and unvaccinated HIV[-] women. We likewise 
will calculate the point prevalence and the prevalence of persisting infection with 95%CI for all 
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HPV types in aggregate and in sub-groups of HPV types according to the protection afforded by 
Gardasil®: Gardasil®-targeted types (HPV6, 11, 16, and 18), Gardasil®-untargeted types for 
which there might be cross-protection (HPV31, 33, and 45) (40, 106, 107), and Gardasil®-
untargeted HPV types for which there is little or no evidence of cross-protection (HPV35, 39, 51, 
52, 53, 56, 58, 59, 66, and 68). Results will be stratified by age and number of doses (3 vs. 2) as 
well as other factors possibly associated with HPV prevalence. Differences in the prevalence of 
these HPV type sub-groups (targeted, possible cross-protection, and untargeted) between the four 
study groups of women will be tested using Fisher’s exact or Pearson chi-square test. Differences 
in the prevalence of these HPV type subgroups by age, number of doses (3 vs. 2), and other 
factors within the group, will be tested for statistical significance (p<0.05) using a Fisher’s exact 
or Pearson chi-square test.
Notably, the study groups of participants are fundamentally different populations (vs. a 
randomized control trial that would recruit from the same population and, as result of 
randomization, enroll similar, representative populations in each arm). Specifically, there are 
known differences in age (and therefore possible differences sexual behaviors) between 
Gardasil®-vaccinated vs. -unvaccinated WLWH, and possible differences in sexual behaviors 
between Gardasil®-vaccinated WLWH vs. HIV[-] women (since HIV infection predominately is 
sexually transmitted in this population). Therefore, we will use a relative measure of 
effectiveness to account for the differences in age and possible differences in exposure to HPV. 
We will use logistic regression to calculate the odds ratio (OR) of the point prevalence and the 
prevalence of persisting HPV infections of Gardasil®-targeted HPV types, individually (HPV6, 
11, 16, or 18) and in aggregate (HPV6, 11, 16, and 18), vs. untargeted HPV genotypes for which 
there is no evidence of cross-protection, for each anatomic site individually (cervix, anus, OR 
oral cavity) and combined (cervix, anus, AND oral cavity). That is, we will compare the ratio 
between study groups of participants as follows: 
NHPV6/11/16/18/NHPV35/39/51/52/53/56/58/59/66/68 [vaccinated WLWH] (Group 1)
NHPV6/11/16/18/NHPV35/39/51/52/53/56/58/59/66/68 [unvaccinated WLWH] (Group 2)
or 
NHPV6/11/16/18/NHPV35/39/51/52/53/56/58/59/66/68 [vaccinated WLWH] (Group 1)
NHPV6/11/16/18/NHPV35/39/51/52/53/56/58/59/66/68 [vaccinated HIV[-] women] (Group 3)
or
NHPV6/11/16/18/NHPV35/39/51/52/53/56/58/59/66/68 [vaccinated HIV[-] women] (Group 3)
NHPV6/11/16/18/NHPV35/39/51/52/53/56/58/59/66/68 [unvaccinated HIV[-] women] (Group 4)
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This will allow us to account for differences in HPV exposure, prevalence, and persistence due 
to differences in age (n.b., prevalence equals incidence * duration; prevalent infections at older 
ages tend to be more persistent ((108)) and sexual behaviors between groups. Additional logistic 
regression models may be used to adjust specifically on other factors, including age, number of 
doses, and sexual behaviors, to account for population differences between study groups. 
Similarly, exploratory analyses will compare the effectiveness of HPV vaccination for cross-
protection types and endpoints of cumulative detection of Gardasil®-targeted HPV types-
associated CIN2+ and/or anal intraepithelial neoplasia grade 2 or more severe (AIN2+) (vs. 
untargeted HPV type-associated CIN2+ and/or AIN2+) between study groups.     
For participants in the existing IeDEA database we will extract HIV specific variables. For those 
who we cannot match to the IeDEA database, will extract HIV specific variables from medical 
records. 
In another exploratory analysis, among WLWH, the presumed route of HIV acquisition (either 
maternal to child or behavioral) will be compared with HPV positivity and, among HPV-positive 
women, HPV viral load.
VII.A.2. Sample Size/Power for Gardasil®-vaccinated vs. unvaccinated WLWH. 
For this analysis, we will make the following assumptions: 1) at least 30% prevalence of HPV 
infection (of any anatomic site) at baseline, 25% of which will be Gardasil®-target HPV types 
and 55% will be untargeted HPV types, 2) a 10% loss to follow-up (LTFU) in at follow up visit, 
and 3) at least 70% of HPV infections persist for 6-12 months We justify an HPV prevalence of 
at least 30% based on 30% prevalence of high-risk HPV infection of the cervix in 30-year-old 
WLWH from our previous U54 study (109). We expect that the HPV prevalence may be higher 
since the prevalence of HPV, like that of other sexually transmitted infections, tends to peak 
about 5 years after the median age of sexual initiation in a population. However, we 
conservatively used 30% to ensure adequate statistical power. We justify our assumption of a 
maximum of 10% follow-up, given our experience working in Rwanda over the last ~20 years. 
We justify 70% of prevalent HPV infections persisting for 6-12 months, given that 36-50% of 
prevalent HPV infections persist 6-months in HIV[-] women (109, 110) and WLWH have an 
impaired immunity to HPV (8, 9). 
Sample size calculation relates to the relative effectiveness measure as discussed. Under above 
assumptions, a sample size of 757 vaccinated and 757 unvaccinated WLWH will provide ≥80% 
power (p=0.05) to detect an OR of ≤0.5 for 6-12 month persistent HPV6/11/16/18 infections, 
relative to HPV35/39/51/52/53/56/58/59/66/68 (but not HPV31/33/45 because of possible cross 
protection), in Gardasil®-vaccinated vs. -unvaccinated WLWH. Consequently, because there 
will be more participants with a prevalent infection than 6-12 month persistent HPV infection 
(because there are no losses to follow-up or HPV viral clearance for which to account), there will 
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be ≥80% power (p=0.05) to detect a OR of ≤0.75 in point prevalence of HPV6/11/16/18 
infection relative to HPV35/39/51/52/53/56/58/59/66/68, in Gardasil®-vaccinated vs. -
unvaccinated WLWH.
VII.B. Specific Aim 2
To quantify, and examine the determinants of, long-term antibody responses to, HPV 
vaccination.
VII.B.1. Analyses. 
Geometric mean titers (GMT) and seropositivity (seroconversion) will be compared between 1) 
HPV-vaccinated WLWH and HPV-vaccinated HIV[-] women, overall and stratified on the 
number of vaccine doses, 2) HPV-vaccinated and unvaccinated WLWH, overall and by the 
number of doses received by the HPV-vaccinated WLWH, and 3) three vs. two doses for HPV-
vaccinated WLWH and 4) HPV-vaccinated and unvaccinated HIV[-] women, overall and by the 
number of doses received by the HPV-vaccinated HIV[-] women. 
Differences in GMT and seropositivity will be tested for statistical significance using the Mann-
Whitney and Fisher’s exact tests, respectively. ANOVA and logistic regression models will be 
used to adjust for/assess the association of other factors (e.g., age at vaccination, age at 
enrollment into the study, number of doses, HIV status [positive vs. negative and current 
antiretroviral therapy, CD4 counts, and HIV viral load] and history [e.g., CD4 nadir], and 
detection of HPV genotypes, etc.) with GMT and seropositivity, respectively.
Among vaccinated and unvaccinated HPV seropositive WLWH, the association between HPV 
antibody titer and presumed route of HIV acquisition (either maternal to child or behavioral) will 
be examined. 
VII.B.2 Sample Size/Power to compare Gardasil®-vaccinated WLWH vs. 
Gardasil®-vaccinated HIV[-] women. 
Using the variance of GMT for HPV antibodies from Einstein et al. (111, 112), a sample size of 
at least 274 will provide 80% power (p=0.05) to detect a 30% difference in GMT between HPV-
vaccinated WLWH and HIV[-] women for a given number of doses. If there is no appreciable 
difference in the titers between those who got 3 or two doses, we can combine those groups with 
different doses. In that case, with a sample size of at least 548, there will be 80% power (p=0.05) 
to detect a 22% difference between HPV-vaccinated WLWH and HIV[-] women. 
VII.C. Exploratory Aim: A Study of 6-12 Month Type-Specific HPV Persistence 
Given the size of the cohort of WLWH (n=1,514 at baseline), the planned follow-up of 
the HPV-positive WLWH, and the laboratory assay capacities that are available and will be 
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developed at the study lab, we have unique opportunity to look at determinants of early, short-
term HPV genotype-specific persistence/clearance in young women. Studies of HPV persistence 
in WLWH, especially in SSA, are limited in number and size (113-120). There is a growing body 
of evidence that an altered CVM (cervicovaginal dysbiosis) is associated with persistent HPV 
infection (86) but there are no published data on its role in HPV persistence in WLWH. There is 
evidence of greater cervicovaginal microbiotic diversity in WLWH compared to HIV[-] women 
(121) and a complex, possible inter-dependence of CVM and HPV natural history (122). Given 
the importance of short-term HPV persistence as a biomarker of cervical-cancer risk (123) as 
well as an endpoint for prophylactic HPV vaccine trials, this is a unique opportunity to 
understand its determinants.
Paired baseline and follow up specimens from participants whose baseline cervicovaginal 
specimen tests HPV positive will have their cervicovaginal microbiome characterized. We will 
examine factors such as baseline and history (if available) of HIV disease status (HIV viral load 
and CD4), the presence of the HPV genotype at other anatomic sites, CVM composition, and 
other risk factors (e.g., sexual behaviors) on whether HPV infection persists vs. clears. 
Contingency tables with Pearson chi-square or Fisher’s exact tests (categorical variables) or 
Kruskal-Wallis test (continuous variables) will be used to identify factors potentially associated 
with HPV genotype persistence (vs. clearance) (p<0.1). Vaginal community state types (CSTs) 
will be assigned to specimens based on hierarchical clustering of the top 20 OTUs, in terms of 
sample sums across all samples within the cohort. Prior to clustering OTUs will be agglomerated 
at the species level. In cases where an OTU could not be resolved down to the species level, the 
next lowest assigned taxonomic label will be used. Clustering was performed using the ward D2 
algorithm using Euclidian distances. We will develop a multivariate logistic regression model 
that incorporates generalized estimating equations (GEE) to calculate odds ratios and 95% 
confidence intervals as a measure of association of these factors with the likelihood of HPV 
genotype-specific persistence (vs. clearance). Factors identified in the univariate analyses will be 
added to the model using both forward and backward selection techniques until a final, 
parsimonious model have been achieved. Future studies will examine predictors, including 
anatomic site-specific microbiome, of anal and oral 6-12 month HPV persistence and the inter-
relationship of 6-12 month HPV persistence between anatomic sites.
VIII. CAPACITY BUILDING AND TECHNOLOGY TRANSFER
This study is funded through a U54 grant from the U.S. National Institutes of Health (NIH). 
Several components of this study require capacity building that will have long-term benefits for 
future research in Rwanda. 
1. Next Generation Sequencing (NGS) technology will be established at the study lab to 
perform cervicovaginal and oral microbiota characterization. This will support future 
investigations of the microbiome in sickness and health. A biobank of cervicovaginal and 
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anal specimens will be created to understand the role of anatomic site-specific microbiota 
on corresponding HPV natural history. More generally, with these capacities we can look at 
the impact of HIV infection on anatomic site-specific microbiota. In addition, the capacity 
for doing NGS will allow locally conducted studies of the human genome and genomic 
testing for personalized medicine in Rwanda. 
2. This study builds upon current ELISA capabilities at the study lab to perform titration of 
plasma antibodies, a skill that can be applied to other studies of vaccine response.
3. The current vaccine records for participants living in Kigali will be migrated into a 
common electronic database, which will allow us to conduct studies more easily on the 
impact of HPV vaccination on outcomes. Importantly, this will allow linkage of HPV 
vaccination status to both the HIV registry that the Rwanda MoH has and the Rwanda 
Cancer Registry, which we re-established to investigate the long-term impact of HPV 
vaccination on cancer incidence in WLWH and HIV[-] women. Through collaborations 
with Rwanda MoH, the HIV registry (TRACnet) will be linked with the cancer registry.
IX. RECRUITMENT AND RETENTION 
IX.A. Community Engagement
The recruitment process will include the community health workers and health center staff 
reaching out to the women, providing education, and registering for an enrollment visit. In 
addition, we have a multi-faceted strategy to engage the community before and during the study. 
IX.B. Benefits to Participation 
Participants who have not previously tested positive for HIV will be provided an opportunity for 
rapid HIV testing with pre- and post-counseling. Because this study is recruiting young women, 
it is very unlikely that they will have precancer, but by participating in this study, their precancer 
will be diagnosed and treated.
IX.C. Barriers to Participation
The main barrier to participation is transportation to the study site, for which all participants will 
be reimbursed. All study procedures and services will also be paid for by the study. This study 
will ask questions about women’s sexual history and reproductive health that might make them 
feel uncomfortable. They have the right to not respond to any question with which they are 
uncomfortable.
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IX.D. Informational Materials
All participant education and informational materials as well as consents and questionnaires will 
be culturally adapted and available in English and Kinyarwanda. We will engage with an 
Advisory board, community health workers, and clinical staff to prepare these materials. All 
questions will be administered in Kinyarwanda by an automated audio interview assistance tool 
to ensure privacy and overcome any literacy barriers. 
IX.E. Planning and Timeline
We are recruiting 3,028 women over a 14- to-16-month recruitment period, with an expectation 
of retaining 90% within the first 6 months-12 months of the study, which defines the main study 
endpoint. In the beginning, we expect that enrollment will take one hour per woman but will 
decrease to 30-45 minutes per participant within a few months as the protocol becomes routine 
for the clinical staff. This has been our experience in doing research in Rwanda for 20 years. For 
a dedicated staff of at least 5 full-time nurses and data entry personnel, and 25% clinician time, 
we believe this to be very feasible. 
We have planned for developing the SOPs, procurement, piloting, staff training, development of 
data collection instruments and systems, and tools for quality control. We are budgeting to staff 
the clinical team and reimbursement for travel of staff and participants for the entire study to 
ensure the timely completion of the study as well as the continuity in the conduct of the study 
and the care of participants. 
IX.F. Recruitment Strategies
Potential participants will be recruited from local health clinics and surrounding communities. 
The study staff will work with five public HIV clinics that participate in the IeDEA program in 
Kigali and the WE-ACTx private HIV clinic. If needed, the staff will work with up to four other 
public health clinics to recruit additional participants. During the period of recruitment at a 
health clinic, WLWH and women who are HIV[-] will be solicited using advertisements 
throughout the entire clinic as well as outreach using community health workers. In addition, 
WLWH who are attending routine appointments at the HIV clinics will learn about the study 
during the group study overview sessions held in the health education room.
IX.G. Retention Strategies
We will rely on the study staff to retain the participants through skills learned from previous 
trainings and experiences. We will monitor enrollment and retention through weekly reports 
generated from Research Electronic Database Capture (REDCap).
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 IX.H. Diversity
The Rwandan staff, which has extensive experience working in this community, is very 
knowledgeable about the population. We will be able to work with and through RD Rwanda and 
RMH, the Advisory board, and the community health workers to ensure that a representative 
population of WLWH and the community surrounding the health clinic is recruited into the 
study. As part of the training for the study, we will make certain that the clinical team recruits 
without bias. 
IX.I. Staff
The staff is already receiving the necessary training during our previous U54 grant 
(5U54CA190163) to conduct this study. However, we are allocating several months for protocol 
development and staff training before enrollment starts. The local staff will be responsible for 
providing most of the clinical services and increasingly so as they get additional training.
X.PROTECTION OF HUMAN SUBJECTS
X.A. Risks to Human Subjects 
X.A.1. Human Subjects Involvement, Characteristics, and Design 
This is an observational study to assess the long-term population effectiveness and immunity of 
the HPV vaccine (Gardasil®) in Rwandan WLWH. The study will compare the cervicovaginal, 
anal, and oral prevalent and 6-12 month persistent HPV6/11/16/18 infections in HPV-vaccinated 
WLWH to those in unvaccinated WLWH and HPV-vaccinated HIV[-] women. We will also 
compare the HPV immune response in HPV-vaccinated WLWH to HPV-vaccinated HIV[-] 
women and the impact of switching from 3 doses to 2 doses of Gardasil® in 2015. Finally, the 
study will investigate the risk factors, including the CVM, for HPV persistence in WLWH. The 
long-term goal is to establish a cohort of WLWH in whom we can examine the long-term 
effectiveness of HPV vaccination in WLWH now and in the future.
The study will include 757 WLWH and 757 HIV[-] women who did receive vaccination (birth 
cohorts 1996 and later) and 757 WLWH who did not receive HPV vaccination (birth cohorts 
before 1996) and 757 HIV[-] women who did not receive vaccination (birth cohorts before 
1996). Women attending collaborating clinics and the surrounding communities will be recruited 
by community health care workers and study staff. All women who are interested in the study 
will meet with a study nurse who will give them an informational flyer and provide a brief verbal 
description of the study and complete the eligibility screener. 
At the enrollment visit, the study nurse will describe the details of the study verbally and women 
will be provided an opportunity to answer any questions. To meet the eligibility criteria, women 
will need to confirm they are 18-28 years old, willing, and physically and mentally able to 
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participate, willing to provide written and signed or thumb printed informed consent, and willing 
to take an HIV test if they are not known to be living with HIV. Women who report to be 
menstruating, pregnant or less than 6 weeks post-partum will be asked to make a later 
appointment. Women will be excluded if they report no previous sexual activity, have a history 
of cervical cancer, treatment for cervical abnormalities after cervix screening, or hysterectomy 
and no longer have a cervix. Because this study has age-specific enrollment goals for WLWH 
and HIV[-] women, once those goals are met, the respective cohorts will be excluded.
All participants must sign and date the most current IRB approved version of the study specific 
informed consent (Appendix II) prior to participating in the study or having any study specific 
procedures. It will be the responsibility of Dr. Fabienne Shumbusho and the coordinator/study 
manager to ensure that participants are consented with the latest approved version of the 
informed consent. The study staff will ensure that all sections of the informed consent that 
require completion by participants are completed.
If a participant does not sign an informed consent prior to a study specific procedure, the 
participant will not be considered eligible and will not be screened or registered.
This study is continuing and leveraging the Einstein-Rwanda consortium and local capacity 
developed in a previous SSA Collaborative HIV and Cancer Consortia (U54) grant. The 
consortium has established capacity to conduct large epidemiological and clinical studies on 
HPV and anogenital cancer, colposcopy, and treatment of high-grade cervical abnormalities, 
high-resolution anoscopy (HRA) and treatment of high-grade anal abnormalities, and laboratory 
assays including the AmpFire assay and DNA extraction, all of which will figure prominently in 
the proposed study. Drs. Gad Murenzi and Fabienne Shumbusho (RD Rwanda and RMH) will 
oversee the study with the lead study nurse Athanase Munyaneza. The recruitment, enrollment 
and follow-up visits will be conducted in coordination with five public clinics that participate in 
the IeDEA program and the WE-ACTx private clinic. If needed, the staff will work with up to 
four other public health clinics to recruit additional participants. The research clinic at RMH will 
provide colposcopy and anoscopy examinations with treatment as well as referral for advanced 
lesions as needed.
X.A.2. Study Procedures, Materials, and Potential Risks 
Most participants will have one study enrollment visit in a private room. Contact information 
will be collected and stored securely in the study database. Participants will provide permission 
to obtain clinical information pertaining to HIV monitoring and treatment as well as HPV 
vaccination history. The visit will consist of a study questionnaire that uses ACASI to ask about 
socio-demographic information and risk factors for HPV including reproductive history and 
sexual behaviors. Participants will have a urine pregnancy test to confirm they are not pregnant. 
Those who are not known to be living with HIV will have a rapid HIV test. A study nurse will 
provide pre- and post-counseling for both pregnancy and HIV tests. All participants will provide 
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a blood draw for anti-HPV16 and HPV18 antibody titer testing. All participants will provide a 
cervicovaginal, anal, and oral specimens for high-risk HPV and HPV6 and HPV11 testing. 
Specifically, participants will provide an oral rinse sample collection by gargling and swishing a 
saline solution for 30 seconds before spitting the rinse into a specimen container. The study nurse 
will help the participant count time and provide the collection. Then, while lying on the exam 
table, the study nurse will collect a specimen taken vaginally (without the use of a speculum) 
using a cervical brush. The study nurse will then collect a sample from the anal canal using a 
scored, Dacron swab. In addition, participants not known to be HIV-infected will have a rapid 
HIV test with pre- and post-counseling. 
We anticipate that approximately 30% of participants will test positive for high-risk HPV and/or 
HPV6/11 on any sample and be invited for a 6-12 month follow-up visit. At that visit, 
participants will provide only specimens from those anatomic sites that were HPV-positive at 
baseline to measure 6-12 month HPV type-specific persistence. For safety purposes, WLWH and 
HIV[-] women with a cervicovaginal type-specific persistent hrHPV infection will be referred to 
colposcopy, and WLWH with an anal type-specific persistent HPV16/18 infection will be 
referred to anoscopy (Section VI.G). At the colposcopy and anoscopy procedure and follow-up 
visits, participants will be treated as needed.
All participant data collected will be entered or transferred to a secure REDCap database, with 
access to personal information restricted to the local staff. Study data will be stored in REDCap 
and maintained on password protected study computers behind the institution firewall. REDCap 
is a secure, web-based application designed to support data capture for research studies 
providing: 1) an intuitive interface for validated data entry; 2) audit trails for tracking data 
manipulation and export procedures; 3) automated export procedures for seamless data 
downloads to common statistical packages; and 4) procedures for importing data from external 
sources.
Potential risks from these procedures include: 
•
Eligibility Screening. Potential participants will be asked to confirm their history of any 
sexual activity and that might make them feel uncomfortable. This question will be asked 
in a neutral manner in a private location.  
•
Urine Pregnancy Test. A pregnancy test can produce anxiety both before and after testing. 
Participants will be asked before testing whether they believe they might be pregnant. If 
they test positive, the study nurse will provide counseling as needed for the unexpected 
result.
•
Rapid HIV Testing. HIV testing can produce anxiety both before and after testing.
•
Cervicovaginal Collection. Collection of cervicovaginal specimens involves a modest risk 
of bleeding and is typically very limited when it occurs. Testing positive for any test may 
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cause psychological distress (anxiety). Excessive bleeding from cervicovaginal specimen 
collection is very rare. 
•
Anal Collection. Collection of anal specimens involves a modest risk of bleeding and is 
typically very limited when it occurs. Testing positive for any test may cause psychological 
distress (anxiety). Excessive bleeding from anal canal specimen collection is very rare. 
•
Oral Collection. Collection of oral specimens involves a modest risk of bleeding and is 
typically very limited when it occurs. Testing positive for any test may cause psychological 
distress (anxiety). Excessive bleeding from the collection of an oral specimen is very rare. 
•
Blood Draw. There is occasionally the possibility of discomfort, bleeding, or bruising from 
the blood draw. 
•
Colposcopy, Biopsies and Digital Cervical Images. Biopsies may induce vaginal bleeding 
and may incur pain, infection, and short-term psychological distress (anxiety). Excessive 
bleeding from a biopsy is rare. A diagnosis of CIN2 or more severe diagnoses may cause 
psychological distress (anxiety). A diagnosis of invasive cervical cancer may cause severe 
psychological distress. 
•
Anoscopy and Biopsies. Participants may feel a cramp of pinch during the biopsy. Many 
people do not feel the biopsy. They may notice a scant amount of bleeding with a bowel 
movement or wiping for a couple days. Most often there are not serious side effects from 
this procedure. Complications such as bleeding or infection are rare but can occur. 
•
LEEP. LEEP may induce vaginal bleeding and may incur pain, infection, and short-term 
psychological distress (anxiety). Excessive bleeding from a LEEP is rare. 
•
85% trichloroacetic acid for small lesions or infrared coagulation (IRC) for larger lesions. 
85% trichloroacetic acid uncommonly may induce local discomfort and pain. For IRC, 
there can be mild to moderate pain or discomfort which can be treated with pain 
medication. Bleeding or discharge with bowel movements can occur for up to 2-3 weeks. 
Rare complications (< 1%) include infection in the anal area and severe bleeding. 
•
Ablative Treatments. Ablative treatments typically induce pain, cramping, and watery 
discharge. Ablative treatment may cause infection, malodorous discharge, and cervicitis. 
Fainting due to a vasovagal reaction from cryotherapy or thermal ablation is rare. Vaginal 
bleeding is rare. Cervical stenosis is rare. 
•
Questionnaire. Participants will be asked questions about sexual history and reproductive 
health that might make them feel uncomfortable. They will be conducted in private, using 
an automated audio interview assistance tool ACASI. 
•
There is also the risk of psycho-social stress that would occur if there was inadvertent 
disclosure of confidential medical or other personal information. All medical and personal 
information will be provided to participants in a private study room to ensure privacy.
•
Women may seek alternative screening and treatment services such as they exist in Kigali 
or elsewhere if they do not wish to participate in the study. 
•
The study investigators and staff will work with the local MoH and clinic staff to ensure all 
study activities will be conducted in compliance with local COVID-19 pandemic 
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guidelines. Necessary precautions will be taken to mitigate the risk of COVID-19 
transmission among study participants and staff. This includes cleaning all tablets and 
headphones used by study participants and staff in between visits. All study staff and 
participants will wear masks and utilize hand sanitizer regularly. All surfaces will be 
frequently cleaned with disinfectant. Whenever possible, windows will be left open for 
ventilation. 
X.B. Adequacy of Protection Against Risks
X.B.1. Informed Consent and Assent 
Women will be recruited during interactions with the community health workers and health 
center staff. During this outreach women will be provided education and allowed to register for 
an enrollment visit. 
At the enrollment visit in the private exam room, the study nurse will describe the details of the 
study verbally and women will be provided an opportunity to ask any questions. The participant 
eligibility will be verified using the eligibility form. Those who qualify and are able and willing 
will then undergo the consenting process, in which the two copies of the consent form, one for 
the study documentation and one for the participant to take home for her records. The consent 
form includes study information such as contact information for study doctors in case of 
questions, concerns, or emergencies. The study nurse and the participant will go through a 
consent form, which will be read by the study nurse to those participants who are illiterate, 
allowing for sufficient time for questions and answers about the procedures and processes. 
All participants must sign and date the most current IRB-approved version of the study 
specific informed consent prior to participating in the study or having any study specific 
procedures. It will be the responsibility of Dr. Fabienne Shumbusho and coordinator/study 
manager to ensure that participants are consented with the latest approved version of the 
informed consent. The study staff will ensure that all sections of the informed consent that 
require completion by participants are completed.
If a participant does not sign an informed consent prior to a study specific procedure, the 
participant will not be considered eligible and will not be screened or registered. A copy of the 
informed consent will be archived in the participant’s research file. The informed consent 
process will be documented in the study log.
X.B.2. Protections Against Risk 
Protection against the risk of inadvertent disclosure of confidential information is addressed by 
the standard procedures at the Rwandan study site, including: (i) storing completed paper copies 
of study forms and other hard copy information (described above), identified by study number 
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only, in a filing system separate from the name-address file of participants in the study; and (ii) 
only the designated local personnel have access to cross-reference the files. 
Copies of signed consents will be stored in locked file cabinets in a locked room, with access 
restricted to study personnel only, at the study lab. Data will be entered or transferred to a secure 
REDCap database, with access to personal information restricted to the local staff. 
Study data will be stored in REDCap and maintained on password protected study computers 
behind the institution firewall. REDCap is a secure, web-based application designed to support 
data capture for research studies providing: 1), an intuitive interface for validated data entry; 2) 
audit trails for tracking data manipulation and export procedures; 3) automated export procedures 
for seamless data downloads to common statistical packages; and 4) procedures for importing 
data from external sources. REDCap is hosted on a secure server and has undergone a 
Governance Risk & Compliance Assessment by All REDCap electronic data files shared with 
Albert Einstein College of Medicine will be maintained by the HIPAA-compliant Epidemiology 
Study Management and Informatics Core Facility (ESMI) at Einstein. The Albert Einstein 
College of Medicine policy on use of REDCap can be found at: 
http://ric.einstein.yu.edu/ric_files/REDCap%20Appropriate%20Use%20Policy.pdf 
Side effects/adverse events due to ablative treatments are generally minor (pain, cramping, and 
watery discharge). Any pain, bleeding, or stress that might occur related to colposcopy, biopsies, 
vaginal collections, and/or LEEP are typically modest and no greater than those risk for the 
primarily HIV[-] women in the general population for whom cervical screening is recommended 
by the WHO and most countries in the world. These side effects and serious adverse events will 
be managed through referral to the Gynecology department at RMH. As needed, consults with 
other medical specialists will be sought. Care for study-related adverse events will be paid for by 
the study. Serious adverse events will be reported to the IRBs.
Study nurses have been trained in pre- and post-counseling for HIV testing and will provide 
support to participants taking rapid HIV tests. They will be trained to assist participants with any 
anxiety or discomfort experienced through responses to questionnaires and/or testing HPV 
positive.
Although women who are pregnant are at very low risk of adverse events due to the enrollment 
and follow-up procedures in this study, it is important to avoid any potential perceived risk. For 
this reason, all participants will have a pregnancy test performed at all visits. If they are found to 
be pregnant, they will be exited from the study and invited to enroll when they are at least 6 
weeks post-partum. 
Risks associated with blood collections will be minimized by having a trained and experienced 
health worker draw blood. 
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Because of the nature of the study, we believe that there is a very low risk of incidental findings 
related to participants’ health and well-being. That said, we will have language in the informed 
consent acknowledging the possibility of incidental findings, and whether and how that 
information will be disclosed confidentially to the individual. Should there be an incidental 
health finding, the information will be provided to the participant if they consented to do so, and 
the research team will ensure that basic educational information about the nature of the finding is 
made available and will assist in finding the appropriate referral to a clinician or specialist for 
care if so desired.
X.C. Potential Benefits of the Proposed Research to Research Participants and Others 
Participants who have not previously tested positive for HIV will be provided an opportunity for 
rapid HIV testing with pre- and post-counseling. Any participant who tests positive for the rapid 
HIV test at enrollment will be connected to the local HIV clinic for evaluation and management. 
Because this study is recruiting young women, it is very unlikely that they will have precancer. 
But by participating in this study, their precancer will be diagnosed and treated. This study will 
generate important information contributing to the knowledge of HPV vaccine effectiveness and 
immunity in Rwanda women living with HIV and without HIV. The findings will help to inform 
the development of programs and interventions for communities.
XI. LIMITATION AND FUTURE DIRECTIONS 
Several limitations in the proposed study are worth noting. Our study is not a randomized trial, 
but the study populations will be selected from different cohorts of women: WLWH who are too 
old to have been vaccinated, WLWH and HIV[-] women who were vaccinated with 3 doses,  
WLWH and HIV[-] women who were vaccinated with 2 doses, and HIV[-] women who were too 
old to have been vaccinated. We have proposed several approaches to account/adjust for those 
age differences but as with any observational study, these techniques are unable to completely 
control for this bias.
We are using 6-12 month persistence as a proxy for cancer risk but ideally, we want to measure 
the impact of Gardasil® on high-grade cervical and anal abnormalities as a more proximal 
surrogate for cancer risk. The population will be still too young to have large numbers of these 
endpoints although we will look at these endpoints in secondary analyses. The longer-term 
effectiveness of Gardasil® through years of peak HPV exposure cannot be assessed in the 5-year 
time period of a single grant.
Our hope is to conduct follow-up studies of this newly established cohort of WLWH to assess 
the durability of immunogenicity and protection, including protection against high-grade cervical 
and anal abnormalities, offered by HPV4 for WLWH, as well as continuing to study the natural 
history of HPV in WLWH living in SSA. Eventually, as noted above (Section VIII.), we will be 
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well-positioned to examine the impact of HPV vaccination on cancer incidence in WLWH by 
linkage of electronic databases that we have helped/will help create.
XII. DISSEMINATION OF RESEARCH RESULTS 
We plan to publish a series of 10 or more scientific reports in peer-review scientific journals. As 
building research capacity in Rwanda is a major goal of this U54 grant, all members of the 
research team will be asked and supported to lead at least one analysis and one manuscript 
preparation, based on interests and expertise. A publication grid is in development to assign 
analytic and publication responsibilities equally and collaboratively among both Rwandan and 
U.S. investigators.
XIII. TIMELINE
Pending approvals and situations in the field with the COVID-19 pandemic, we anticipate 
starting enrollment in September 2021. Enrollment will take approximately 14-16 months with 
completion of follow-up visits 6-12 months after that. All colposcopy and treatment visits should 
be completed by mid-2023.
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APPENDIX I. VISUAL SUMMARY OF THE STUDY PROTOCOL
Enrollment Visit
•
Cervical, anal, oral specimens for HPV typing
•
Blood for HPV serology
Recruit and Consent
•
757 HPV-vaccinated WLWH
•
757 unvaccinated WLWH
•
757 HPV-vaccinated HIV[-] women
•
757 unvaccinated HIV[-] women
HPV-negative for
cervicovaginal, anal
and oral specimens
Exit
Cervicovaginal, anal,
and/or oral HPV+*
•
Cervicovaginal specimens: no
type-specific persistent HPV
•
Anal specimens: for WLWH, no
persistent HPV16 or HPV18
Follow-up Visit
•
Repeat cervicovaginal, anal, or
oral specimens for HPV typing
Exit
•
Cervicovaginal specimens: type-
specific persistence of high-risk
types
•
Anal specimens: for WLWH,
persistent HPV16 or HPV18
Colposcopy and/or HRA
•
Cervical images taken at
colposcopy for algorithm
development
Cervical and/or anal treatment
for high-grade abnormalities
*high-risk HPV or HPV6/11 positive
APPENDIX II. WRITTEN INFORMED CONSENT DOCUMENT
KEY INFORMATION FOR LONG-TERM HPV VACCINATION EFFECTIVENESS AND IMMUNITY IN 
RWANDAN WOMEN LIVING WITH OR WITHOUT HIV
We are asking you to choose whether or not to volunteer for a research study about how well the 
HPV vaccine works for women living in Rwanda. This page is designed to give you key 
information to help you decide whether to participate. We have included detailed information 
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after this page. Ask the research team questions. If you have questions later, please contact the 
research investigator in charge of the study. Their contact information is below.
WHAT IS THE STUDY ABOUT AND HOW LONG WILL IT LAST? 
Cancer of the cervix (the opening of the womb), anus, and throat can be caused by a virus – the 
human papillomavirus, also called HPV. We know that when the HPV vaccine is given to girls, 
it prevents infection from certain types of HPV. We want to understand how the vaccine prevents 
HPV infection for women many years after they were vaccinated. We also want to understand 
whether the vaccine works differently for women with HIV. By doing this study, we hope to 
learn among women like yourself in Rwanda, how well the vaccine works in the future. Your 
participation in this research will last about one year. Today, you will be asked to conduct a short 
questionnaire, provide a blood sample, and provide 3 samples of cells collected by a study nurse 
(vaginal, oral, and anal samples). If you do not have HIV, you will have an HIV test today. If any 
of these samples have HPV, we will ask you to return in 6-12 months for additional visits so you 
can provide samples again and see if you still have HPV or if it has gone away on its own. If you 
still have HPV, we may ask you to see a special doctor to make sure you do not have precancer. 
REASONS YOU MIGHT CHOOSE TO VOLUNTEER FOR THIS STUDY
You will be adding to the knowledge of how HPV vaccination works in the future for women 
with different health conditions in Rwanda. Your participation will specifically help us 
understand how to improve HPV vaccination for women who live with HIV. For a complete 
description of benefits, refer to the Consent Document below.
REASONS YOU MIGHT CHOOSE NOT TO VOLUNTEER FOR THIS STUDY
We do not anticipate any mental, emotional, or physical harm through participation in this study. 
However, you may feel concerned about the confidentiality of the information shared as part of 
this research. Your responses will be kept completely confidential. None of the information you 
provide will be shared with anyone outside of the study team.
DO YOU HAVE TO TAKE PART IN THE STUDY?
Your participation is voluntary. You are being asked to complete the survey, but you may choose 
not to do so. You may choose to skip any questions that you do not wish to answer. If you sign 
the consent form document below, you have agreed to become part of the research study.
If you decide to take part, you are free to stop participating at any time without giving a reason. 
However, some of the information may have already been entered into the study and that will not 
be removed. The researchers may continue to use and share the information they have already 
collected.
WHAT IF YOU HAVE QUESTIONS, SUGGESTIONS OR CONCERNS?
The person in charge of the study is Gad Murenzi, MD, MPH. If you have questions, 
suggestions, or concerns regarding this study or you want to withdraw from the study. His 
contact information is: 
Gad Murenzi MD, MPH, Principal Investigator
RD Rwanda, Kigali, Rwanda 
Tel: +250 788 589 085 / Email: gadcollins@gmail.com
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If you are unable to reach Dr. Murenzi, Dr. Fabienne Shumbusho, MD is another researcher 
responsible for this study that you can reach out to. Her contact information is:
Dr. Fabienne Shumbusho, MD
RD Rwanda, Kigali, Rwanda 
Tel: +250 788 559 065 / Email: fabienneshumbusho@gmail.com
As the study will be conducted in Rwanda, if you wish to ask questions about the study or your 
rights as a research participant to someone other than the researchers or if you wish to voice any 
problems or concerns you may have about the study, please contact the chairperson of the 
Rwanda National Ethics Committee (RNEC) Dr. Mazarati Jean Baptiste at (250) 78830987 or 
Dr. Tumusiime David at (250) 788749398 or by mail: info@rnecrwanda.org.
If you have any additional questions, suggestions, or concerns about your rights as a volunteer in 
this research, contact staff in the Einstein Institutional Review Board (IRB) at 
irb@einstein.yu.edu.
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RWANDA NATIONAL ETHICS COMMITTEE/ALBERT EINSTEIN COLLEGE OF 
MEDICINE 
DOCUMENTATION OF INFORMED CONSENT AND HEALTH INSURANCE 
PORTABILITY AND ACCOUNTABILITY ACT OF 1996 (HIPAA) AUTHORIZATION
Introduction
You are being asked to participate in a research study called “Long-Term HPV Vaccination 
Effectiveness and Immunity in Rwandan Women Living with and without HIV”. Your 
participation is voluntary -- it is up to you whether you would like to participate. It is fine to say 
“no” now or at any time after you have started the study. If you say “no,” your decision will not 
affect any of your rights or benefits or your access to care. 
Before you decide to be part of this research study, you need to understand the risks and 
benefits so that you can make an informed decision. This is known as informed consent. This 
consent form provides information about the study, which will also be explained to you. Once 
you understand the study and the risks involved, you will be asked to sign this form, or in the 
case that you are illiterate, provide your thumbprint on it and have it signed by a witness, if you 
want to take part. Remember, it is your decision: participation in the study is entirely voluntary.
If you cannot read, a study staff will help you read and understand the written paper that you 
will sign or thumbprint in the presence of a witness you trust, should you decide to join the 
study.
The researcher in charge of this project is called 
the “Principal Investigator.” His name is Gad 
Murenzi, MD, MPH. You can reach Dr. 
Murenzi by phone at (250) 788589085. 
Fabienne Shumbusho, MD will be serving as a 
Co-Investigator of this study, and she can be 
reached by phone at (250) 0788559065. They are 
both researchers at:
Research for Development (RD Rwanda) and 
Rwanda Military Hospital 
Kigali, Rwanda
For questions about the research study, or if you 
believe you have an injury, contact the Principal 
Investigator, Co-Investigator, RNEC or the 
Einstein IRB.
Support for this research study is provided by the 
National Institutes of Health (USA) through 
Grant No: 6U54CA190163-06.
The Rwanda National Ethics Committee 
(RNEC) and Institutional Review Board (IRB) 
of the Albert Einstein College of Medicine 
have approved this research study. The IRB # 
is in the stamp in the upper right-hand corner 
and bottom right corner as well.
If you have questions regarding your rights as 
a research subject, you may contact RNEC 
chairpersons Dr. Mazarati Jean Baptiste at 
(250) 78830987 or Dr. Tumusiime David at 
(250) 788749398. You can also reach the 
RNEC by email at info@rnecrwanda.org or by 
mail:
Rwanda National Ethics Committee 
Ministry of Health
P.O.Box.84
Kigali, Rwanda
If you have any additional questions, please 
feel free to contact the Einstein IRB at 
irb@einstein.yu.edu. 
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Why is this study being done? 
HPV infection is responsible for most of cancer of the cervix (the opening of the womb), 
especially for those women with HIV infection. Some other less-common cancers found in the 
anus and the throat are also caused by HPV. For over 10 years, a vaccine for HPV has been 
given to older girls in Rwanda. We know that when the HPV vaccine is given to girls, it prevents 
infection from certain types of HPV. We want to understand how the vaccine prevents HPV 
infection for women many years after they were vaccinated. We also want to understand whether 
the vaccine works differently for women with HIV. By doing this study, we hope to answer these 
questions.
Why am I being asked to participate? 
You are being asked to take part in this study because you are a woman, aged 18-28 years of age, 
physically and mentally able and willing to participate in the study, and able and willing to 
provide written, informed consent, or else consent with a thumbprint.
What will happen if I participate in the study? 
Today, in a private location at the study clinic you will complete a short questionnaire about your 
life, your reproductive health, and your sexual history. All your answers are strictly confidential. 
You do not have to respond to any question that makes you uncomfortable. You will provide a 
urine sample for a pregnancy test to confirm you are not pregnant. If you do not have HIV, you 
will take a rapid HIV test with a finger prick. A study nurse will provide counseling for you to 
understand what the results mean for you. A study nurse will collect a sample of your blood into 
a collection tube for testing on how your body responds to the HPV vaccine and possible 
infection. In a private exam room, the study nurse will help you swish a saline solution in your 
mouth and then spit it into a container. To collect cells from your vagina, the study nurse will 
insert a soft brush into your vagina, turn it three times and remove it. To collect cells from your 
anal canal the study nurse will insert a small swab into your anal canal, turn it for 20 seconds and 
remove it. Both collections are quick procedures with minimal discomfort. If all three samples do 
not have HPV, you are done with the study visits. If any of these three samples has HPV, we will 
ask you to return in 6-12 months for another visit so you can provide samples again and see if 
you still have HPV or if it has gone away on its own. If you still have HPV, we may ask you to 
see a special doctor to make sure you do not have precancer. At your visit with the special 
doctor, they will take one or more digital pictures of your cervix with a digital camera. The 
picture will not show the outside of your body, just the inside where the cervix is. Please note 
that the pictures will not identify you, and that picture of a cervix will not show your face or 
genitals. People cannot tell the specific woman from whom the cervix picture is taken. All 
pictures will be shown to you at the time if you want to see them. 
How many people will take part in the research study?
You will be one of approximately 3,028 women participating in this study. We will be enrolling 
1,514 women living with HIV and 1,514 women who are not infected with HIV. 
Will there be audio and/or video recording?
No, there will be no audio or video recording in this study. You will hear an audio recording 
for the questionnaire that will be completed in private.
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Information Banking (Future Use and Storage)
●
No testing of human genes will be conducted as part of the current study.
●
ADDITIONAL TESTS ON YOUR SAMPLE: No other tests other than those 
explained under this study are currently planned.
However, if you agree, the specimens you provide for this research may be stored for future 
research testing. The specimen cannot be linked to you. In the future, researchers can apply for 
permission to use the specimen for new studies to prevent, diagnose or treat disease. Future 
testing may require samples to be analyzed in Rwanda, the United States, or elsewhere for 
testing. Your specimens/data may be kept for a long time, perhaps longer than 50 years. 
If you agree to the future use, some of your de-identified specimens, digital pictures of your 
cervix and health information (not linked to you) may be placed into one or more scientific 
databases. Your specimens may also be submitted to a tissue/cell/DNA bank. You have the 
right to withdraw consent to use of the tissue for future use at any time by contacting the study 
doctors listed above. If that is your request, your unused specimens will be destroyed at the 
end of this study. Some researchers may develop tests, treatments or products that are worth 
money. You will not receive payment of any kind for your specimens/data or for any tests, 
treatments, products, or other things of value that may result from the research.
You can choose not to participate in the tissue/cell/DNA bank and still be part of the main 
study and this will not affect your treatment at this facility.
PLEASE INDICATE YOUR CHOICE BY INITIALING ONE (1) OF THE FOLLOWING 
OPTIONS FOR EACH QUESTION
1. Specimens 
_____ I consent to have my specimens stored and used for future research studies.
_____ I do NOT consent to have my specimens stored and used for future research studies. The 
specimens will be destroyed at the end of the study.
2. Future Contact About This Study 
____I consent to be contacted with general information about this study’s research findings.
____I do NOT consent to be contacted with general information about this study’s research 
findings.
3. Future Research
___I consent to being contacted about future research opportunities.
___I do NOT consent to being contacted about future research opportunities.
Will I be compensated for being in this research study? 
You will receive a total of 10,000 RWF in cash to compensate for your time and transport costs 
during the study visit and other follow-up visits if necessary. If you choose to withdraw from the 
study before all components of your participation are completed, you will be compensated only 
for the components you completed.
Will it cost me anything to participate in this study? 
There will be no cost to you to participate in the study.
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Confidentiality
The researchers and study staff follow US federal, US state, and Rwandan laws to protect your 
privacy. This part of the consent form tells you what information about you may be used and 
shared in the research described in this form. You do not have to sign this form but, if you do 
not, you may not participate in the research. 
The health information that we may use or disclose for the research described in this form 
includes information from your entire medical record, such as your name, phone number, email, 
medical diagnoses, dates, test results, social security number, medical record numbers, etc. In 
addition, the researchers wish to review information from your medical records pertaining to 
your HIV status and previous clinical care. If you are living with HIV or test positive for HIV 
today, the study staff may review information about your current and previous HIV care and 
laboratory tests from the health clinic. By law, you must specifically authorize access to these 
records: 
☐ Yes, I authorize the use and disclosure of my information pertaining to HIV testing and HIV 
status. 
Initial: ________ 
Date: ________
Your information and research records will be kept confidential. Your study information will be 
kept if they are useful for the research described in this form. The only people who can see your 
research records are:
●
Researchers and other individuals who work with the researchers
●
Organizations and institutions involved in this research, including those that fund the 
research, if applicable
●
Groups that review research such as central reviewers, Institutional Review Boards, the 
Office for Human Research Protections, the US Food and Drug Administration, data 
coordinating centers, and domestic and foreign agencies that regulate research.
The purposes of these uses and disclosures are to (1) conduct the study and (2) make sure the 
study is being done correctly. The information covered under this form may no longer be 
protected by federal privacy laws (such as HIPAA) once disclosed, and those persons who 
receive your health information may share your information with others without your additional 
permission. All these groups are required to keep your information confidential. 
Certificate of Confidentiality
As a way to protect your privacy, we have obtained a Certificate of Confidentiality from the 
National Institutes of Health, which is funding this study. If information from this study were 
requested or subpoenaed by government agencies or the courts, we would use the Certificate to 
attempt to legally refuse to provide that information. These requests are rare – in only a few 
cases did researchers have to use the Certificate, and it was honored most of the time, but not 
every time. There are several kinds of situations to which the Certificate does not apply. For 
example, we are still required to report child abuse and some diseases, and we must make data 
available to the government for review or evaluation of our research. The Certificate does not 
prevent you or a member of your family from voluntarily sharing information. Similarly, if an 
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insurer, employer, or other person obtains your written consent to receive research information, 
then the researchers may not use the Certificate to withhold that information.
Are there any risks to me? 
We do not think there are any physical risks related to participating in this research study. We 
will ask you questions about your sexual history and reproductive health that might make you 
feel uncomfortable. You have a right not to respond to any question that makes you 
uncomfortable.
A risk of taking part in this study is the possibility of a loss of confidentiality or privacy. Loss of 
privacy means having your personal information shared with someone who is not on the study 
team and was not supposed to see or know about your information. The study team plans to 
protect your privacy – see the Confidentiality section above for details.
However, there is occasionally the possibility of discomfort, bleeding, or bruising from the blood 
draw. These risks will be minimized by having a trained and experienced health worker draw 
your blood.
Are there possible benefits to me? 
Unless you are known to have HIV, you will have a rapid test and learn whether you have HIV. 
It is very unlikely that you have cervical or anal precancer. By participating in this study, any 
precancer diagnosed would be treated. More likely, you will not experience any direct benefit 
personally from participating in this study. We hope you will participate because the study will 
generate important information contributing to the knowledge of HPV vaccine effectiveness and 
immunity in Rwanda women living with or without HIV. Your participation will help to inform 
the development of programs and interventions for communities of people like yourself. 
Will I get to know the results from this study?
If you have an HIV test, you will learn those results immediately. The result of the HPV 
infection test performed on you will not be communicated directly to you. However, if any of the 
three samples become positive, you may be contacted for follow-up visits approximately six-
twelve months later. And if, at follow-up visits, the HPV results remain positive, the study team 
may perform further tests to rule out precancer or refer you to the nearest specialized health 
service if there is any advanced disease. Some other results, which are obtained for research 
purposes only, will not be directly returned to you. However, the overall results of the research 
study will be made available to the public.
What choices do I have other than participating in this study?
You can refuse to participate in the study. If you decide not to participate, the medical care 
providers at this facility will still give you all standard care and treatment that is recommended 
for you.
Are there any consequences to me if I decide to stop participating in this study?
No. If you decide to take part, you are free to stop participating at any time without giving a 
reason. This will not affect your care and you will continue to be treated at this facility. 
However, some of the information may have already been entered into the study and that will not 
be removed. The researchers and the sponsor may continue to use and share the information they 
have already collected.
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To withdraw your consent and authorization, you must contact the Principal or Co-Investigator 
in writing at the address on page 1 of this form. However, you may first call or speak to the 
Principal or co-Investigator and he/she will stop collecting new information about you. If you 
take back your consent and authorization, you will not be allowed to continue to participate in 
this research study. 
Who can answer my questions about the study?
You can talk to the research team member that explains the study to you about any questions, 
concerns, or complaints. You may also telephone Dr. Gad Murenzi / Dr. Fabienne 
Shumbusho on 0788589085/0788559065. If you wish to ask questions about the study or your 
rights as a research participant to someone other than the researchers or if you wish to voice any 
problems or concerns you may have about the study, please contact: RNEC chairperson Dr. 
Mazarati Jean Baptiste at (250) 78830987 or Dr. Tumusiime David at (250) 788749398 or by 
mail: info@rnecrwanda.org
Consent
You have been given a copy of this consent form.
Declaration
CONSENT TO PARTICIPATE
I have read the consent form and I understand that it is up to me whether I participate. I know 
enough about the purpose, methods, risks, and benefits of the research study to decide that I want 
to take part in it. I understand that I am not waiving any of my legal rights by signing this 
informed consent document. I will be given a signed copy of this consent form.
_________________________ ________________________________________
_________
Printed name of participant
Signature or thumbprint of participant
Date
_________________________ ________________________________________
_________
Printed name of the person 
conducting the consent process
Signature
Date
If the person is unable to read and/or write, an impartial witness should be present during the 
informed consent discussion. After the informed consent form is read and explained to the 
person, and after they have orally consented to their participation in the study and have either 
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signed the consent form or provided their fingerprint, the witness should sign and personally date 
the consent form. By signing the consent form, the witness attests that the information in the 
consent form and any other written information was accurately explained to, and apparently 
understood by, the person and that informed consent was freely given by the person.
________________________
  _______________________________________
Printed name of witness if participant 
utilized a thumbprint to consent to participation 
in this study
Signature of witness
Date
_________________________
____________________________________
____
________
_
Printed name of the person conducting the 
consent process
Signature
Date
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APPENDIX III. ENROLLMENT QUESTIONNAIRE
Thank you for agreeing to participate in our study. This interview should take no more than 20 
minutes. Some of these questions are personal about your life, your reproductive health, and your 
sexual history. All your answers are strictly confidential, and it is very important for us that you 
try to answer all the questions as openly as possible, even if they are a little uncomfortable or 
embarrassing. This information will be important in understanding how the HPV vaccine 
protects women against HPV infection. If you don’t understand any of the questions, please 
inform the study nurse who will try to help explain them. You should also be aware that you 
have a right not to respond to any question with which you are uncomfortable.
A. Socio-demographics
1. What is your date of birth? DD/ MM /YYYY (enter all 0 if not remembered) 
_ _ / _ _ / _ _ _ _
2. What is your age? _ _ (Year)
3. What is your marital status?
❑ Married/Cohabiting
❑ Divorced
❑ Widowed 
❑ Separated 
❑ Single
❑ Choose not to answer
4. Do you live in Kigali?
❑ Yes
❑ No (Skip to Question #6)
❑ Choose not to answer (Skip to Question #6)
5. In what area in Kigali City Province do you live?
❑ Nyarugenge
❑ Kicukiro
❑ Gasabo urban (e.g., Kacyiru, Kimironko, Kinyinya, etc.) Gasabo rural (e.g., Nduba, 
Jabana, Gikomero, etc.)
❑ Do not know
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❑ Choose not to answer
6. Who do you live with? (Check all that apply)
❑ Live alone
❑ Partner/spouse
❑ Daughter(s)/son(s)
❑ Parents and/or other family
❑ Other: _________________
7. What is your primary occupation?
❑ Student
❑ Employed by government, another institution, or company
❑ Self-employed (Small and medium enterprises) 
❑ Self-employed (High income earnings)
❑ Unemployed/Do not work
❑ Other (specify): ______________________  
❑ Choose not to answer (Skip to Question #8)
The following questions are sensitive and personal in nature. Your answers will be kept 
confidential. You may choose not to answer certain questions. Answering any question is 
voluntary
B. Parity 
8. What age did you have your first full term birth ? _ _ (Year) (Enter 00 if refuse to answer, 
enter 99 if never pregnant or not yet had a pregnancy that resulted in a full term birth – i.e. all 
pre-term deliveries) (if 00 or 99, skip to Question #11)
9. How many full term births have you had in your lifetime? ________ 
10. Have you given birth in the last year?
❑ Yes
❑ No
❑ Choose not to answer
C. Contraceptive Use
11. Have you ever used Jadell (contraceptive)?
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❑ Never
❑ No (Skip to Question #13)
❑ Choose not to answer (Skip to Question #13)
12. Do you currently use Jadell (contraceptive)?
❑ Yes
❑ No
❑ Choose not to answer
13. Have you ever used oral contraceptives?
❑ Yes
❑ No (Skip to Question #15)
❑ Choose not to answer (Skip to Question #15)
 14. Do you currently use oral contraceptives?
❑ Yes
❑ No
❑ Choose not to answer
15. Have you ever used Depo Provera (contraceptive)?
❑ Yes
❑ No (Skip to Question #17)
❑ Choose not to answer (Skip to Question #17)
16. Do you currently use Depo Provera (contraceptive)?
❑ Yes
❑ No
❑ Choose not to answer
17. Do use condoms as a contraceptive?
❑ Yes
❑ No (skip to Question #19)
❑ Choose not to answer (skip to Question #19)
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18. Have you used condoms as a contraceptive in the last 6 months?
❑ Yes
❑ No
❑ Choose not to answer
19. Have you ever used another contraceptive method?
❑ Yes. Please specify: _____________________
❑ No (skip to Question #21)
❑ Choose not to answer (skip to Question #21)
20. Do you currently use another contraceptive method?
❑ Yes. Please specify: _________________________
❑ No
❑ Choose not to answer
D. Sexual Behaviors 
This last section of the questionnaire contains questions about sexual practices you may have 
engaged in over the years. This will help us understand your likelihood of having been exposed 
to human papillomavirus (HPV).
21. How many men have you had vaginal sex with, in your lifetime until now? Vaginal sex is a 
man inserting his penis into your vagina.
❑ No Men (Skip to question #25)
❑ 1 Man
❑ 2-3 Men
❑ 4-5 Men
❑ 6 or more Men
❑ Choose not to answer
22. How old were you when you first had vaginal sex? _ _ (Year) (Enter 00 if refuse to answer)
23. How many men have you had vaginal sex with in the last 6 months? 
❑ No Men (Skip to Question #25)
❑ 1 Man
❑ 2 or more Men
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❑ Choose not to answer
24. In the past 6 months, how often has he used a condom during vaginal intercourse?
❑ Never 
❑ Occasionally
❑ Half the time
❑ Almost always 
❑ Always
25. Have you ever had anal intercourse? Anal intercourse is a man inserting his penis into your 
anus/rectum.
❑ Yes
❑ No (Skip to Question #29)
❑ Choose not to answer (Skip to Question #29)
26. How many men have you had anal sex with, in your lifetime until now?
❑ 1 Man
❑ 2-3 Men
❑ 4-5 Men
❑ 6 or more Men
❑ Choose not to answer
27. How many men have you had anal sex with, in the past 6 months?
❑ No Men (Skip to Question #29)
❑ 1 Man
❑ 2 or more Men
❑ Choose not to answer
28. In the past 6 months, how often has heused a condom during anal sex?
❑ Never 
❑ Occasionally
❑ Half the time
❑ Almost always 
❑ Always
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29. Have you ever performed oral sex on a man? (Oral sex would be a blow job or you sucking 
someone’s penis)
❑ Yes
❑ No (Skip to Question #32)
❑ Choose not to answer (Skip to Question #32)
30. In your lifetime until now, how many men have you performed oral sex on until now?
❑ 1 Man
❑ 2-3 Men
❑ 4-5 Men
❑ 6 or more Men
❑ Choose not to answer
31. During the past 6 months, how many men have you performed oral sex on?
❑ No Men
❑ 1 Man
❑ 2 or more Men
❑ Choose not to answer
E. HPV Vaccination History
32. Have you had the HPV vaccine (most women have the vaccine when they are around 11-13 
years old)?
❑ Never vaccinated (End of Questionnaire)
❑ Vaccinated 
❑ I don’t remember (End of Questionnaire)
33. How many times have you had the HPV vaccine?
❑ One time
❑ Two times
❑ Three times
❑ I don’t remember
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APPENDIX IV. FOLLOW UP QUESTIONNAIRE
Thank you for agreeing to participate in our study. This interview should take no more than 10 
minutes. These questions are personal about your sexual history. All your answers are strictly 
confidential, and it is very important for us that you try to answer all the questions as openly as 
possible, even if they are a little uncomfortable or embarrassing. This information will be 
important in understanding how the HPV vaccine protects women against HPV infection. If you 
don’t understand any of the questions, please inform the study nurse who will try to help explain 
them. You should also be aware that you have a right not to respond to any question with which 
you are uncomfortable.
1. What is your date of birth? DD/ MM /YYYY (enter all 0 if not remembered) 
_ _ / _ _ / _ _ _ _
2. What is your age? _ _ (Year)
The following questions are sensitive and personal in nature about sexual practices you may have 
engaged in since your last study visit. This will help us understand your likelihood of having 
been newly exposed to human papillomavirus (HPV). Your answers will be kept confidential. 
You may choose not to answer certain questions. Answering any question is voluntary.
3. Since your last visit, how many men have you had vaginal sex with? 
❑ No Men
❑ 1 Man
❑ 2 or more Men
❑ Choose not to answer
4. Since your last visit, how many men have you had anal sex with?
❑ No Men
❑ 1 Man
❑ 2 or more Men
❑ Choose not to answer
5. Since your last visit, how many men have you performed oral sex on?
❑ No Men
❑ 1 Man
❑ 2 or more Men
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❑ Choose not to answer
END OF QUESTIONNAIRE
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1.
Rwanda Population-Based HIV Impact Assessment.  October 2019.
2.
IGCO. GLOBOCAN 2018 database. 2018.
3.
Cervix Cancer Screening. IARC. 2005;10.
4.
Kitchener HC, Castle PE, Cox JT. Chapter 7: Achievements and limitations of cervical 
cytology screening. Vaccine. 2006 Aug 31;24 Suppl 3:S3/63-70. PubMed PMID: 16950019. 
Epub 2006/09/05.
5.
Bruni L, Albero G, Serrano B, Mena M, Gómez D, Muñoz J, et al. Human 
Papillomavirus and Related Diseases in Rwanda. ICO/IARC Information Centre on HPV and 
Cancer (HPV Information Centre) 2019.
6.
Centers for Disease Control and Prevention. 1993 revised classification system for HIV 
infection and expanded surveillance case definition for AIDS among adolescents and adults. 
JAMA. 1993;269(6):729-30.
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1993 revised classification system for HIV infection and expanded surveillance case 
definition for AIDS among adolescents and adults. MMWR Recomm Rep. 1992;41:1-19.
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Grulich AE, van Leeuwen MT, Falster MO, Vajdic CM. Incidence of cancers in people 
with HIV/AIDS compared with immunosuppressed transplant recipients: a meta-analysis. 
Lancet. 2007 Jul 7;370(9581):59-67. PubMed PMID: 17617273. Epub 2007/07/10.
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Silverberg M, al e. Cumulative Incidence of Cancer Among Persons With HIV in North 
America: A Cohort Study. Ann Intern Med. 2015;163(7):507=18.
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Colon-Lopez V, MS S, Machin M, al e. Anal Cancer Risk Among People with HIV 
Infection in the United States. J Clin Oncol. 2018;36(1):68-75.
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World Health Organization. Estimated number of people living with HIV, 2016 By WHO 
region 2017 [Available from: 
http://gamapserver.who.int/mapLibrary/Files/Maps/HIV_all_2016.png.
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HIV Country Intelligence: HIV Country Profiles 2018 [Available from: 
http://cfs.hivci.org/.
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The Global HIV/AIDs Epidemic 2019 [Available from: https://www.kff.org/global-
health-policy/fact-sheet/the-global-hivaids-epidemic/.
14.
Negin J, Cumming RG. HIV infection in older adults in sub-Saharan Africa: 
extrapolating prevalence from existing data. Bull World Health Organ. 2010 Nov 1;88(11):847-
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53. Negin, J., Cumming, R., (2010) HIV infection in older adults in sub-Saharan Africa. PubMed 
PMID: 21076566. PMCID: PMC2971515. Epub 2010/11/16.
15.
PEPFAR. Rwanda Population-Based HIV Impact Assessment (RPHIA 2018-2019) 2019 
[Available from: https://phia.icap.columbia.edu/wp-content/uploads/2019/10/RPHIA-Summary-
Sheet_Oct-2019.pdf.
16.
Schiffman M, Castle PE, Jeronimo J, al e. Human papillomavirus and cervical cancer. 
The Lancet. 2007:890-907.
17.
Schiffman M, Clifford G, Buonaguro FM. Classification of weakly carcinogenic human 
papillomavirus types: addressing the limits of epidemiology at the borderline. Infect Agent 
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