Protocol

Protocol for: Luetkemeyer AF, Donnell D, Dombrowski JC, et al. Postexposure doxycycline to prevent bacterial
sexually transmitted infections. Engl J Med 2023;388:1296-306. DOI: 10.1056/NEJMoa2211934

This trial protocol has been provided by the authors to give readers additional information about the work.
 1

Supplement

This supplement contains the following items:

1. Original protocol, final protocol, summary of changes.
2. The original statistical analysis plan, which is the only version.
 2

Table of contents

Original Protocol 3

Final Protocol 33

Summary of changes to the protocol 71

Statistical Analysis Plan 73

 3

PROTOCOL

DoxyPEP: Evaluation of doxycycline post-exposure prophylaxis to reduce sexually transmitted infections in

PrEP users and HIV-infected men who have sex with men

Version 1.0

January 31, 2019

 4

Table of Contents

STUDY TEAM ................................................................................................................................................... 4

GLOSSARY....................................................................................................................................................... 5
SCHEMA ........................................................................................................................................................... 6
1.0 BACKGROUND AND SIGNIFICANCE ................................................................................................ 7
1.1 Background ............................................................................................................................................. 7
1.2 Rationale ................................................................................................................................................. 9
2.0 STUDY DESIGN ................................................................................................................................ 11
3.0 OBJECTIVES .................................................................................................................................... 12
3.1 Primary Objectives............................................................................................................................. 12
3.2 Secondary Objectives ........................................................................................................................ 12
3.3 Exploratory objectives........................................................................................................................ 12
4.0 SELECTION AND ENROLLMENT OF PARTICIPANTS ........................................................................ 12
4.1 Inclusion criteria .................................................................................................................................... 12
4.2 Exclusion criteria ............................................................................................................................... 12
4.3 Recruitment ....................................................................................................................................... 13
5.0 STUDY TREATMENT ....................................................................................................................... 13
5.1 Study product ........................................................................................................................................ 13
5.2 Safety of doxycycline ............................................................................................................................. 13
5.3 Doxycline dispensing and administration. .............................................................................................. 14
5.3 Concomitant Medications ....................................................................................................................... 14
5.3.1 Prohibited medications .................................................................................................................... 14
5.3.2 Precautionary Medications .............................................................................................................. 14
5.4 HIV pre-exposure prophylaxis (PrEP) and HIV antitroviral therapy (ART) .............................................. 14
6.0 CLINICAL AND LABORATORY MONITORING ......................................................................................... 16
6.1 Schedule of Events ................................................................................................................................ 16
6.2 Study visits ............................................................................................................................................ 17
6.3 Instructions for evaluations .................................................................................................................... 17
6.4 Management and treatment of GC, CT and syphilis .............................................................................. 19
6.5 Testing for resistence in setting of STI diagnoses on study ................................................................... 19
7.0 ADVERSE EVENTS (AE) AND STUDY MONITORING ..................................................................... 20
7.1 Adverse Event Collection Requirements ............................................................................................ 20
7.2 AE and SAE attribution to doxycycline ............................................................................................... 20
7.3 Study Monitoring ................................................................................................................................ 20
7.3.1 Study Team Monitoring ................................................................................................................... 21
7.3.2 Independent Monitor ....................................................................................................................... 21
7.3.3 DSMB ............................................................................................................................................. 21
 5

8.0 CLINICAL MANAGEMENT ISSUES .................................................................................................. 21

8.1 Toxicity .............................................................................................................................................. 21
8.2 Specific Management of Toxicities Related to Study-Provided Drugs ................................................ 22
9.0 CRITERIA FOR DISCONTINUATION................................................................................................ 22
9.1 Premature Study Treatment Discontinuation...................................................................................... 22
9.2 Premature Study Discontinuation....................................................................................................... 23
10.0 STATISTICAL CONSIDERATIONS ................................................................................................... 23
10.1 Outcome measures.............................................................................................................................. 23
10.2 Statistical power and analysis .............................................................................................................. 23
10.3 Endpoint adjudication.......................................................................................................................... 24
12.0 PARTICIPANTS ................................................................................................................................ 24
12.1 Institutional Review Board (IRB) Review ........................................................................................ 24
12.3 Study records................................................................................................................................. 25
13.0 BIOHAZARD CONTAINMENT ........................................................................................................... 25
 6

STUDY TEAM

Connie Celum, MD, MPH, co-Principal Investigator

Annie Luetkemeyer, MD, co-Principal Investigator
Jared Baeten, MD, PhD
Ruanne Barnabas, MBChB, DPhil
Susan Buchbinder, MD, MPH
Katerina Christopoulos, MD, MPH
Edwin D. Charlebois, MPH, PhD
Stephanie Cohen, MD, MPH
Julie Dombrowski, MD, MPH
Deborah Donnell, PhD
Diane Havlir, MD
Chaz Langelier, MD,PhD
Hyman Scott, MD, MPH
Olusegun Soge, MSc, PhD
 7

GLOSSARY

AE Adverse event
AMR antimicrobial resistance
ART Antiretroviral therapy
CT Chlamydia trachomatis
GC Gonorrea
HIV Human Immunodeficiency virus
MSM Men who have sex with men
NAAT Nucleic acid amplification test
PEP Post-exposture prophylaxis
PLWH People living with HIV
PrEP Pre-exposure prophlaxis
RPR Rapid Plasma Reagin
SAE Serious Adverse Event
STI Sexually transmitted infection
TCN tetracycline
VDRL Venereral Disease Research Lab
 8

SCHEMA

DoxyPEP: Prospective, randomized, open-label, clinical trial to evaluate the impact of doxycycline
post-exposure prophylaxis on the occurence of bacterial STIs among MSM PREP users and HIV-
infected men who have sex with men

Design: Open-label randomized clinical trial of doxycyline PEP to reduce bacterial STIs (N.
gonorrheae (GC), C. trachomatis (CT), and T. pallidum (syphilis) among MSM persons living
with HIV (PLWH) and HIV-uninfected MSM on PrEP.

Study Population: 390 PWLH participants and 390 HIV-uninfected participants, for a total sample size of 780

Study Sites:
1. HIV/AIDS Clinic (“Ward 86”), Zuckerberg San Francisco General Hospital, UCSF, San
Francisco
2. San Francisco City Clinic, San Francisco Department of Public Health, San Francisco
(municipal STD clinic)
3. Madison HIV/AIDS Clinic, Harborview Medical Center University of Washington, Seattle
4. Public Health-Seattle & King County STD Clinic at Harborview Medical Center, Seattle
Primary Study Objectives:
1. Evaluate the effectiveness of doxycycline post-exposure prophylaxis (PEP) to reduce
STI infections in two populations: a) MSM living with HIV and b) HIV-uninfected MSM
taking HIV PrEP
2. Investigate the impact of doxycycline PEP on development of culture-based tetracycline
(TCN) resistance in N. gonorrheae and among nasopharyngeal carriers of S. aureus

Secondary Study Objectives:

1. Assess the safety, tolerability, and acceptability of doxycycline PEP
2. Investigate the impact of doxycycline PEP on development molecular markers of
tetracycline (TCN) resistance C. trachomatis (CT), and T. pallidum (syphilis).
3. Evaluate the development of phenotypic tetracycline resistance among nasopharyngeal
carriers of commensal Neisseria species.
4. Measure the coverage of sex acts by doxycycline PEP
5. Assess the effect of doxycycline PEP on the gut resistome through measuring the
abundance of tetracycline resistance genes in a subset of 50 HIV-infected MSM and 50
HIV-uninfected MSM assigned to receive doxycycline PEP as well as rectal swabs from
all participants, stored for future evaluation.

Approach: Eligible participants randomized to receive PEP will receive open-label doxycycline 200 mg
to be taken ideally within 24 hours but no later than 72 hours after each condomless sexual
contact (receptive anal, insertive anal, vaginal, or oral, or use of sex toys). No more than
200 mg will be taken in each 24 hour period. Sexual activity and doxycycline PEP usage will
be recorded using a smartphone application and assesement at study visits. At enrollment
and quarterly for 12 months of follow-up, GC and CT testing will be conducted on samples
from the oropharynx, rectum and urine and a blood specimen for syphilis serology will be
collected. Resistance testing will be conducted on specimens that test positive for GC using
phenotypic methods, and molecular techniques for CT and syphilis (for syphilis, DNA
obtained from mucosal or lesional swabs). Nares and pharyngeal swabs will be obtained to
 9

evaluate tetracycline resistance among nasopharyngeal carriers of S. aureus and

commensal Neisseria species. Stool samples from 50 MSM living with HIV and 50 HIV-
uninfected MSM on PrEP in the doxycycline PEP arm at 3 time points will undergo
metagenomic sequencing for tetracycline resistance genes. Rectal swabs from all
participants who consent will be archived for future evaluation of the enteric microbiome and
resistome.

Study Schema

1.0 BACKGROUND AND SIGNIFICANCE

1.1 Background
The incidence of bacterial sexually transmitted infection (STI) reached a new high in the US in 2016, with more
than 2 million diagnoses of syphilis, gonorrhea (GC) and chlamydia (CT).4 This STI epidemic is occurring globally
and is concentrated among men who have sex with men (MSM), including those living with and without HIV. In
the US, MSM accounted for 81% of syphilis infections in 2016 and 38% of gonorrhea cases; the latter represents
a nearly 10-fold increase since 1989. 4 CT is also increasing among MSM, with rectal chlamydia rates of 17%
and the highest rates of CT diagnosed in STI clinics in MSM ≥ 25 years old.4 Both MSM living with HIV and
HIV-uninfected MSM on PrEP have very high bacterial STI incidence – with the prevalence of new STI diagnoses
50% per year or higher.5, 6

Increasing STI rates have been associated with a rise in serious morbidity, including blindness secondary to
syphilitic ocular complications.7 Congenital syphilis rates have increased 28% in the past few years;8 it is likely
that some of this increase comes from ‘bridging’ from MSM to heterosexual populations.9 Distressingly, drug-
resistant GC continues to rise globally10-12 and in the US,13 leading to limited oral treatment options; recent cases
of highly-resistant GC in the UK and Australia required treatment with ertapenem.14, 15 Untreated STIs increase
the risk of HIV transmission from HIV-infected individuals who are not virologically suppressed,16, 17 as occurs in
nearly half of those living with HIV nationwide.18 With the recent CDC-endorsed “U=U” campaign (i.e.,
undetectable=untransmittable),19 condom use may further decrease among HIV-infected persons, and may
impact ‘serosorting’ in selection of sexual partners and decisions to use condoms. Accordingly, there is an urgent
 10

need for innovative, effective , and acceptable interventions to halt the rise in syphilis, GC, and CT among MSM
living with and at-risk for HIV infection.

One potential STI control strategy for these high-risk populations is post-exposure prophylaxis (PEP)
with doxycycline. Antibiotic prophylaxis against STIs is not a new concept; minocycline was used as PEP for
GC in the US Navy in the 1940s and showed transient benefits with no clinical harms, but the rapid emergence
of tetracycline resistance prevented uptake of this approach as a public health strategy 20 A systematic review
of presumptive periodic treatment of sex workers showed overall 50% reductions in GC and CT without risk
compensation or increased AMR, and this approach is recommended for populations with high baseline GC and
CT prevalence 21. The rationale for STI prophylaxis among MSM is similar, given the high prevalence and
incidence of bacterial STIs among MSM living with HIV and MSM on PrEP. An effective STI prophylaxis strategy
could augment frequent STI screening, partner notification and other efforts to reduce STI acquisition and
transmission among MSM.

Doxycycline has good tolerability, high bioavailability with oral Figure 1. IPERGAY Study: Time to first STI (panel a), GC
dosing, a 20 hour half-life, and excellent tissue penetration. infection (panel b), CT infection (panel c), and syphilis
infection (panel d) 1
Doxycycline is a first line treatment for CT with very high efficacy
(97-100%). Doxycycline is used to treat syphilis for persons with
penicillin allergy. Notably, CT and syphilis have not developed TCN
resistance in spite of decades of doxycycline use to treat these
infections. Doxycycline is not recommended for GC treatment due
to baseline resistance and increased TCN resistance will not affect
GC management.

The IPERGAY study demonstrated efficacy of single dose

doxycycline PEP after condomless sex among 232 HIV-negative
MSM taking event-driven PrEP in France.22 Doxycycline PEP
resulted in a 47% relative reduction in new bacterial STIs (GC, CT,
or syphilis) over 8.7 months of follow-up (45.9 vs. 79.6 per 100
person-years in those assigned to doxycycline PEP vs. no PEP
control, respectively; Figure 1 panel a). Doxycycline PEP reduced
the incidence of CT and syphilis, but not GC (Figure 1, panels b-d),
which was not surprising given doxycycline resistance in >50% of
GC strains in France. MSM in IPERGAY had a median age 38,
were highly educated, the majority were Caucasian, and highly
adherent to event-driven HIV PrEP. MSM in the doxycycline arm
in IPERGAY took a median of 680 mg of doxycycline per month.
Adverse events were rare and there was no difference in self-
reported sexual behavior between MSM in the two study arms.

Limitations of generalizability from the IPERGAY study include that

the sample was small (n=232), and in contrast to MSM in the US
heavily impacted by STIs, MSM were older (median age 38), highly educated, and majority were Caucasian. In
addition with respect to adherence to event-driven doxycycline PEP, MSM in the IPERGAY study were highly
adherent to event-driven HIV PrEPand were already ‘cueing’ their TDF-FTC PrEP dosing to sexual activity.
Importantly, uptake and adherence to doxycycline PEP among daily PrEP users are unknown. Additionally,
IPERGAY did not conduct detailed measurements of the pattern of doxycycline use with sexual practices nor
evaluate for antibiotic resistance associated with intermittent doxycycline use. Doxycycline PEP may be more
efficacious for reducing GC incidence in the US than in Europe, due to lower GC TCN resistance in the US (25%
vs 55% respectively).13, 22 PEP may also be more effective to prevent GC at non-pharyngeal sites, as evidenced
by no effect on pharyngeal GC incidence whereas there was a trend toward reduction in rectal and urethral GC
infections with doxycycline PEP in IPERGAY.23
 11

A small study piloted daily doxycycline among 30 MSM living with HIV with a history of syphilis, and showed a
73% reduction in composite STIs, without power to assess individual STIs.24 The greater risk of side effects and
antimicrobial resistance (AMR) with daily dosing of doxycycline have dampened enthusiasm for daily dosing of
doxycycline as PrEP against STIs. Event-driven doxycycline PEP has not been studied in MSM living with HIV.

Significant scientific knowledge gaps remain about on-demand STI doxycycline PEP in PrEP users and
HIV-infected MSM. MSM in IPERGAY were already ‘cueing’ their Truvada PrEP dosing to sexual activity; PEP
adherence in daily PrEP users is unknown. IPERGAY did not conduct detailed measurements of the pattern of
doxycycline use with sexual practices nor report antibiotic resistance associated with intermittent doxycycline
use. Doxycycline PEP may be more efficacious for reducing GC incidence in the US than in Europe, due to lower
TCN resistance in GC the US (30% vs 55% respectively).13, 22 PEP may also be more effective to prevent GC at
non-pharyngeal sites; there was a trend toward reduction in rectal and urethral infections with doxycycline PEP
in IPERGAY.23 Notably, based on the IPERGAY demonstration of efficacy with doxycycline PEP, providers have
begun to prescribe doxycycline PEP to reduce incident STIs in MSM without a confirmatory study or normative
guidance. Moreover, the balance of benefits and risks of doxycycline PEP need to be carefully studied in MSM
living with HIV as well as in PrEP users in a study powered to assess effectiveness for each population. The
relative benefits and risks of doxycycline PEP may differ in MSM living with HIV and PrEP users due to
differences in adherence, sexual practices, potential risk compensation, sexual networks, and/or pre-existing
antibiotic resistance due to higher prior antibiotic exposure among HIV-infected MSM.

In summary, a sufficiently-powered, high-quality study is needed to evaluate the effectiveness of

doxycycline PEP in both HIV-infected and HIV-uninfected populations, and to evaluate its impact on drug
resistance in target bacterial STIs and resident bacterial microbiota. There is a public health need and
sufficient clinical equipoise to conduct an effectiveness study of doxycycline PEP as an STI reduction strategy
for a diverse population of MSM in the US to address the following key scientific questions: 1) Effectiveness of
doxycycline PEP for bacterial STI prevention in MSM on daily PrEP, 2) Effectiveness of doxycycline PEP in MSM
living with HIV; 3) Differential tolerability and impact on antimicrobial resistance between PLWH and HIV-
uninfected MSM on PREP; 4) TCN resistance in GC, CT and syphilis, 5) TCN resistance in commensal Neisseria
(which can transfer TCN resistance) and pathogenic Staph Aureus in carriers, and 6) effect on the gut resistome
in terms of changes in in TCN resistance genes.

1.2 Rationale
The overall goal of the proposal is to understand the effectiveness of doxycycline PEP on reducing STIs in MSM
populations at high risk for STI acquisition and to evaluate its impact on antibiotic resistance.

1.2.1 Doxycycline Safety

Several features of doxycycline make it an optimal choice for STI PEP. Doxycycline has been used safely and
for extended durations as prophylaxis against infections such as malaria25 and for non-infectious conditions
including rosacea26 and acne vulgaris.27 Doxycycline is generally safe and well-tolerated when used chronically.
Side effects include mild gastrointestinal symptoms, photosensitivity, and rarely pill esophagitis and pseudotumor
cerebri.28 Reassuringly, doxycycline has a much lower risk than other antibiotics for the antibiotic-associated
diarrhea caused by C. difficile infection,29, 30 and may even exert a protective effect against C. difficile.31, 32
Doxycycline is a scalable PEP intervention, as it is generic, inexpensive, and widely available. Doxycycline does
not require dose adjustment in hepatic or renal failure, including dialysis. In the IPERGAY doxycycline PEP
study, there was no difference in grade 3 or 4 adverse events between the study arms. As anticipated, there
were more gastrointestinal side effects in those on PEP (25%) vs. the non-PEP control arm (14%).
Discontinuation of doxycycline was uncommon with 8(7%) stopping doxycycyline due to side effects attributed
to doxycycline, the majority of which were gastrointestinal.1 Given the safety profile of doxycycline and the
enrollment of participants receiving ongoing clinical care for HIV or HIV PrEP, study-specific monitoring will be
limited to complete blood count and liver function tests at months 6 and 12 to assess for hematologic and hepatic
 12

toxicity, which occur rarely (<1%). Assessment for side effects (eg., pill esophagitis, photosensitivity, new onset
headaches), with clinical and laboratory testing if indicated if potential serious adverse events are suspected.
Participants will receive standard of care monitoring through their HIV or PrEP. Typical standard of care
monitoring for PLHIV includes semi-annual viral load monitoring, renal and liver function tests, and annual CBC.
Typical standard of care monitoring for PrEP patients includes quarterly HIV tests and semi-annual or annual
renal monitoring. .

1.2.2 Rationale for single dose doxycycline as STI PEP.

A concern about a single post-sex dose of doxycycline is the long in vivo doubling time of syphilis (approximately
30-33 hours)33, 34, which is the rationale for treatment of early syphilis with long-acting IM penicillin or oral
doxycycline for 14 days. However, the incubation time for syphilis is 10-90 days,35and doxycycline PEP
administered within 72 hours of exposure may deliver sufficient chemoprophylaxis to abort primary infection.
Notably, in IPERGAY, a 73% reduction in syphilis incidence was observed with single dose doxycycline.1 In
addition, single dose doxycycline PEP has been shown to be effective for Lyme disease36 and leptospirosis 37, 38
– spirochetal diseases that also have slow doubling times.37, 39 An alternative approach is doxycycline PrEP (i.e.,
daily dosing).24 However, daily doxycycline PrEP may have a higher frequency of intolerability due to side
effects, selection of antimicrobial resistance through higher exposure, and greater costs than doxycycline PEP.
Importantly, MSM in IPERGAY demonstrated their ability to effectively adhere to peri-coital Truvada PrEP and
post-coital doxycycline PEP.

1.2.3 Potential for drug resistance

An important consideration with intermittent doxycycline use for STI prophylaxis is selection of drug resistance,
in both target bacterial STI pathogens as well as in microbiota which are potential pathogens or possible
reservoirs of antimicrobial resistance, such as S. aureus and commensal Neisseria species. GC has sequentially
developed resistance to antibiotics in multiple classes, including those previously or currently recommended for
treatment. Rates of TCN-resistance in GC in the US are 25%-38% in studies of MSM,13 compared with >50%
in Europe.22 Doxycycline is not currently recommended as treatment for GC. IPERGAY has not reported on TCN
resistance in GC and did not address AMR in other bacterial such as S. aureus and commensal Neisseria
species. Doxycycline is commonly used to treat CT and syphilis, and there have been no documented cases of
TCN-resistant CT or syphilis. However, TCN resistance from single point mutations has been reported in related
organisms such as Chlamydia suis40, 41 and Brachyspira spp.42 in antibiotic-exposed pigs, suggesting that
development of TCN resistance could evolve (or be acquired from a resistant organism such as E. coli 43).
Azithromycin-resistant Treponema pallidum emerged from a single mutation44 and rapidly became widespread
in the US west coast and globally,45 serving as a cautionary tale.

Repeated antibiotic exposure can foster drug resistance in the normal commensal flora of the body and impact
the composition of gut microbiome. The effect of doxycycline use on S. aureus is an important consideration,
given that ∼30% of the population are S aureus carriers.46 AMR is concerning for community-based MRSA
infections, for which doxycycline is sometimes used as an alternative to sulfa drugs.47 Similarly, doxycycline
exposure could also drive development of TCN resistance in commensal Neisseria species, serving as a
reservoir that potentially can transfer resistance to GC. Antibiotics affect the larger microbiome, with decreased
diversity of gut flora and shifts in the predominant species, which may fuel a “dysbiotic” environment and may
contribute to inflammation and complications such as C. difficile.48-50 In sum, a comprehensive assessment of
doxycycline PEP is needed, including public health benefits, safety, tolerability, adherence, and AMR among
bacterial STIs and organisms that may become pathogens (e.g. S. aureus among carriers) or transfer resistance
to GC (e.g., from commensal Neisseria).

1.2.4 Rationale for evaluation of meningococcal vaccination status

A recent report from New Zealand suggests that vaccination with an outer membrane-based Group B
Neisseria meningitidis vaccine is associated with a reduced risk of gonorrhea in adolescents and adults aged
15-30 years of age; the estimated effectiveness of the OMV meningococcal vaccine against gonococcal
 13

infection to be 33% [ NZ OMV is part of the composition of the 4CMenB vaccine, commercially distributed as
BEXSERO51 (GSK). It is not known if other meningococcal vaccines are associated with a similar decrease in
GC infections. Meningococcal vaccination (Serogroup A/C/W/Y) is currently recommended by the ACIP for all
PLWH and serogroup B vaccination is a consideration for adolescents and young adults52. Meningococcal
vaccine status will be recorded, with the type of vaccine if known, to evaluate for a potential association of
vaccination with decreased GC rates.

2.0 STUDY DESIGN

Design: The overarching goal is to assess the effectiveness of doxycycline PEP on incidence of STIs and
tetracycline resistance among STIs and commensal bacteria to inform public health policy. Participants will be
randomized 2:1 (see Schema), with a greater number receiving doxycycline PEP compared with the standard of
care control, to maximize data on safety, tolerability, adherence coverage of sexual acts, and resistance data in
participants randomized to doxycyline PEP, without negatively impacting power to measure effectiveness.
Participants will be counseled about the preliminary effectiveness data from IPERGAY, and the potential for
AMR in STIs or other bacteria. Possiblity of unreported doxycycline us in the control arm (contamination) will be
monitored through retrospective batch testing of doxycycline metabolites in hair, to detect doxycycline use in the
prior 3 months. 53

The trial of doxycyline PEP will be powered to separately assess impact for MSM living with HIV and HIV-
uninfected MSM on PrEP because of potential differences in safety, tolerability, adherence, sexual networks,
sexual practices, background AMR, and ultimately, PEP effectiveness. Eligible participants randomized 2:1 to
receive PEP will receive open-label doxycycline 200 mg to be taken ideally within 24 hours but no later than 72
hours after condomless sexual contact (oral or anal). 200 mg of doxycycline will be taken at most once per 24
hour period regardless of the number of sexual acts occurring during this time period. he rationale for 2:1
randomization is to maximize data on doxycycline PEP safety, tolerability, acceptability, and resistance, while
providing sufficient power for the primary outcome of doxycycline PEP effectiveness. Sexual activity will be
recorded for both arms of the study (doxycycline PEP and control condition) by the participant using a
smartphone application that will be adapted for study use; this will enable comparable assessment of risk in the
two arms. PEP pill-taking will also be measured by the app to enable assessment of coverage of sex acts by
PEP. Sexual activity and adherence will also be assessed in person at quarterly visits. STI testing will be
conducted quarterly from three anatomic sites (pharyngeal, rectal and urinary) and blood for syphilis testing.
Participants with a positive STI test will return for STI treatment and for swabs of the affected site for resistance
testing; culture based for GC and molecular methods for CT and syphilis. Those with a serologic test that
indicates a new syphilis infection will have swabs of any current active lesion as well as mucosal swabs from the
orophanynx. Nares and oropharyngeal swabs will be obtained at baseline, 6 and 12 months to evaluate
tetracycline resistance in S. aureus among carriers and in commensal Neisseria species.

Stool samples from 100 participants on the doxycycline PEP arm − 50 MSM living with HIV and 50 HIV uninfected
MSM on PrEP− will be collected at baseline, 6 and 12 months to evaluate effects of intermittent doxycycline on
the gut resistome, using 16s ribosomal RNA amplification to study tetracycline resistance genes. Rectal swabs
will be collected and archived in all participants at baseline, 6, and 12 months for future studies of the impact of
doxycycline PEP on the enteric microbiome and resistome.

Study population: This study will enroll 390 HIV-infected participants and 390 persons taking PrEP, for a total
sample size of 780. An approximately equal number of participants in each of these cohorts (and in each study
arm) will be enrolled in San Francisco and Seattle.

Current or planned initiation of PrEP use is an eligibility criterion for enrollment, because this population of MSM
has high rates of STIs and are typically seen quarterly for PrEP visits. However, participants may opt to stop
PrEP use at any time during the study without affecting their involvement in the study. Any HIV-uninfected
 14

participants who subsequently seroconvert will be managed clinically by the study site according to local practice
(appropriate counseling, clinical evaluation and immediate linkage to clinical and psychosocial services). These
participants will also be retained in the study unless they choose to discontinue study participation.

3.0 OBJECTIVES
3.1 Primary Objectives

3.1.1 Evaluate the effectiveness of doxycycline post-exposure prophylaxis (PEP) to reduce STI infections
in two populations: a) MSM living with HIV and b) HIV-uninfected MSM taking HIV PrEP.
3.1.2 Investigate the impact of doxycycline PEP on development of culture-based tetracycline (TCN)
resistance in N. gonorrheae and among nasopharyngeal carriers of S. aureus

3.2 Secondary Objectives

3.2.1 Assess the safety, tolerability, and acceptability of doxycycline PEP
3.2.2 Measure the coverage of sex acts by doxycycline PEP
3.2.3 Evaluate the development of TCN-resistance among nasopharyngeal carriers ofcommensal
Neisseria species
3.2.4 Assess the effect of doxycycline PEP on the gut resistome through measuring the abundance of
tetracycline resistance genes in a subset of 50 HIV-infected MSM and 50 HIV-uninfected MSM

3.3 Exploratory objectives

3.3.1 Evaluation the diversity of the gut microbiome and quantity and breadth of drug resistance genes in
participants on PEP and not on PEP

3.3.2 Evaluate the association between meningococcal vaccination status and incident GC infection

4.0 SELECTION AND ENROLLMENT OF PARTICIPANTS

4.1 Inclusion criteria

4.1.1 Willing and able to give written informed consent

4.1.2 Age > 18 years
4.1.3 Male sex at birth
4.1.4 Previously HIV-diagnosed
OR
HIV-seronegative at the time of last test within the past month and a current prescription for PrEP
(both daily or event-driven permitted) or plan to start PrEP within 30 days after the enrollment visit
4.1.5 Condomless anal or oral sexual contact with ≥ 1 male sex-at-birth partners in the past 12 months
4.1.6 Diagnosed with GC, CT or early syphilis (primary, secondary or early latent) in the past 12 months.
Note: self report of STI is acceptable if documentation not available.
4.1.7 Willing to use a smartphone app to record sexual practices (all participants) and doxycycline PEP (if
assigned to PEP arm) if the participant possesses a smartphone
Note: Possession of smartphone is not required for study participation. Study personnel will assist in
obtaining a smartphone through locally available resources if participant does not have a phone.

4.2 Exclusion criteria

4.2.1 Allergy to tetracycline class

4.2.2 Current medications which may impact doxycycline metabolism or that are contraindicated with
doxycycline, as per the prescribing information. These include systemic retinoids, barbiturates,
carbamazepine, and phenytoin.
 15

4.2.3 Anticipated use of doxycycline during the coming 12 months for non-STI prevention (e.g., acne
treatment).

4.3 Recruitment
Each site has established local recruitment and screening methods that operationalize protocol-specified
requirements for eligibility determination in a manner that is tailored to and most efficient for the local study
setting and target study population. Each site will use a variety of recruitment approaches, including direct
recruitment at clinics, referrals from other providers of PrEP and ART, and use of online and social networking
websites and apps. Recruitment materials will educate men about STI prevalence and incidence in their
community and the pilot study that demonstrated efficacy of doxycycline for reducing incidence of STIs among
MSM in IPERGAY.

The study will enroll from 4 clinical sites: 2 in Seattle and 2 in San Francisco (Table 1), which have established
track records of high quality research in MSM populations integrated into clinical care settings; annual retention
rates in site clinical trials on average exceed 80%-90%. The sites have large MSM populations that are
ethnically and racially diverse.

Table 1. Study Sites

High volume, comprehensive HIV care clinics with • “Ward 86” HIV Clinic, ZSFG, UCSF, San
on-site STI testing & treatment. Francsico, CA
• University of Washington Harborview Madison
HIV clinic, Seattle, WA
Municipal STI clinics with integrated on-site HIV • San Francisco City Clinic, San Francisco CA
PrEP programs. • Harborview STD Clinic/Public Health-Seattle &
King County, Seattle, WA

5.0 STUDY TREATMENT

5.1 Study product

Generic immediate release doxcycyline monohydrate 100 mg capsule (Mayne Pharmaceuticals) will be
provided by the study to participants randomized to open-label PEP. Doxycycline monohydrate is a standard
formulation of doxycycline that is widely avaible and FDA approved for the treatment of a number of infectious
conditions. The full prescribing information can be accessed at
https://dailymed.nlm.nih.gov/dailymed/getFile.cfm?setid=54ce5887-5e72-4d7a-a821-
936dd3ffa68c&type=pdf&name=54ce5887-5e72-4d7a-a821-936dd3ffa68c

5.2 Safety of doxycycline

Doxycycline is an antibiotic that can be used for prolonged periods of several months at a dosage of 100 mg /
day in the treatment of acne or in the prevention of malaria. The tolerance of repeated-dose of doxycycline in
this 12 months study should therefore be acceptable.

The most common side effects of doxycycline are54, 55

• Gastrointestinal: nausea, diarrhea, epigastric pain, anorexia, glossitis, enterocolitis, anal and genital
candidiasis
• Esophageal disorders: dysphagia, pill esophagitis, and rarely esophageal ulceration. Risk for
esophageal irritation can be reduced by taking doxycycline tablets at least one hour before bedtime (to
avoid being lying down with doxycycline intake) at a meal with a large glass of water (100 ml).
 16

• Skin reactions including maculopapular, erythematous and photosensitivity skin reactions. Due to
concen for photosensitivity, individuals taking doxycycline are advised to avoid excessive exposure to
sunlight and UV radiation (such as tanning beds).

Other possible but rare adverse effects occurring in < 1% in post marketing reports include:
• Hypersensitivity reactions: urticarial, anaphylaxis, serum sickness, drug reaction with eosinophilia and
systemic symptoms (DRESS)
• Skin toxicity: fixed drug eruption. Stevens-Johnson syndrome, toxic epidermal necrolysis, erythema
multiforme, fixed drug eruptions exacerbation of preexisting lupus erythematosus
• Pericarditis
• Hematological disorders: hemolytic anemia, thrombocytopenia, neutropenia, eosinophilia
• Intracranial hypertension (also known as pseudotumor cerebri)

5.3 Doxycline dispensing and administration.

Doxycycline will be dispensed at each 3 month visit or in shorter intervals per patient prefence, with additional
dispensation as needed to replace lost medication or provide additional medication when needed. Capsules
should be stored at room temperature (59-86o F) to the extent feasible. Participants will be instructed to take 2
pills of 100 mg doxycycline as soon as possible after condomless sexual contact, ideally within 24 hours but no
later than 72 hours after sex. No more than 200 mg should be taken in a 24 hour period. For particiants reporting
multiple sexual encounters, 200 mg of doxycycline will be recommended to be taken every 24 hours until after
the last sexual encounter. Study staff will instruct participants on procedures for replacement of lost medication.

Doxycycline may be taken on an empty stomach or with food, and it is advised to take with a large glass of water
and to taking at least 60 minutes before bed to reduce risk of esophageal irritation. All participants randomized
to PEP will be advised to avoid excessive sun exposure and artificial UV light, such as tanning beds, and to
consider use of sunscreen if they will be exposed to sun.

5.3 Concomitant Medications

Participants randomized to doxycycline PEP should report current and new medications to the study team to
ensure no concern for drug interactions with doxycycline. The prescribing information for doxycycline
monohydrate should be reviewed to ensure no potential for drug interactions.

5.3.1 Prohibited medications

• Barbiturates
• Carbamazapine
• Phenytoin
• Methoxyflurane
• Systemic retinoids, such as acitretin and isotretinoin

5.3.2 Precautionary Medications

• Warfarin –increased oral anticoagulant effect and hemorrhagic risk, requiring more frequent
monitoring of prothrombin levels and INR surveillance.
• Antacids containing aluminum, iron, calcium, or magnesium, or bismuth subsalicyclate products
(such as Pepto-Bismol) may impair doxycycline absorption and should be separated 2 hours
before or after doxycycline
• Oral iron supplements may impact doxycycline absorption. Iron should be taken more than 2
hours if possible from doxycycline

5.4 HIV pre-exposure prophylaxis (PrEP) and HIV antitroviral therapy (ART)
 17

PrEP and ART will not be provided by the study but will be accessible through local providers following the
standard of care. Each of the participating sites has a clinical infrastructure in place to assist individuals with
obtaining ART and PrEP, including for those without insurance or whose insurance status changes.
 18

6.0 CLINICAL AND LABORATORY MONITORING

6.1 Schedule of Events

Month ( +/- 4 week window)
Month 0 3 6 9 12
Informed consent √
Smartphone app instructions √
Review of current medications √ √* √* √* √*

Antibiotic & STI history √ √ √ √ √

Meningococcal vaccination history √ √
Sexual behavior questionnaire √ √ √ √ √
Dispense pills/adherence counseling* √* √* √* √*
Doxycycline pill count and adherence √* √* √* √*
recall*
Doxycycline PEP acceptability survey* √* √*
STI symptom screen √ √ √ √
General symptom assessment √ √ √ √
Condoms/lubricant √ √ √ √ √
Risk reduction counseling √ √ √ √ √
GC/CT testing (throat & rectal swab,
√ √ √ √ √
urine collection)
Syphilis testing √ √ √ √ √
Swabs/urine at time of confirmed STIs
at time of confirmed STI
for resistance testing
Nasopharyngeal swabs for Staph
√ √ √
Aureus culture
√ (if baseline
Nasopharyngeal swabs for cultures of
√ swab (+) for
Neisseria species
Neisseria
Stool sample from first 50 MSM PLWH &
√* √* √*
first 50 HIV- MSM on PrEP*
Rectal swabs for storage, all participants √ √ √
Collection of hair for doxycycline testing √ √ √ √
HIV testing data from local care (HIV Ab,
√ √ √ √ √
HIV RNA, CD4)
CBC and liver function tests* √* √*
Reimbursement √ √ √ √ √
*intervention arm only
 19

6.2 Study visits

Specific study procedures are detailed in Table 6.1. Visits will take place at enrollment and quarterly thereafter,
up to 12 months. Interim visits may be scheduled for diagnosis and treatment of STI and collection of additional
specimens or for evaluation of possible doxycycline associated adverse events . Participants may also request
interim STI testing.

6.2.1 Enrollment Visit After confirming eligibility through pre-screening and prior to study procedures, we will
obtain written informed consent and collect locating information; the latter will be updated at each study
visit. At enrollment, demographic, behavioral, and clinical information will be collected and a release of
infomration obtained to permit access to medical records. STI testing for chlamydia and gonorrhea through
nucleic acid amplification testing (NAAT) of clinician or self-collected pharyngeal and rectal swabs and first
void urine will be conducted. Serologic testing for syphilis will be conducted. If required STI testing has
been conducted within past 14 days, these results may be used for the enrollment evaluation. Participants
diagnosed with an STI at baseline will be treated; presence of a baseline STI will not preclude enrollment
but STI’s present at time of enrollment will not count towards study endpoints. All STIs will be reported to
the local health department as required per local standard procedures.

Randomization At enrollment, participants will be randomized in a 2:1 ratio to open-label doxycycline PEP
or the standard of care control condition (regular STI counseling, screening and treatment as indicated).
The randomization code and resulting allocation list will be generated and maintained by the study
statistician. The list will be blocked and stratified by site. While neither participants nor study staff will be
blind to each participant’s randomization group once assigned, in order to protect the blind prior to
randomization of each participant, the randomization scheme will utilize varying block sizes.

6.2.2 Quarterly visits after enrollment. Quarterly visits will be conducted every 90 days, +/- 4weeks. Participants
unable to attend a quarterly visit should be asked to return for an unscheduled visit as soon as feasible.
Subsequent visit should occur on the assigned quarterly scheduled to the extent possible.

6.2.3 Unscheduled interim visits: Participants will be instructed to contact the study site and return for an
unscheduled visit for any of the following a) new diagnosis of STI, b) concern for possible adverse event
associated with doxycycline, c) need for additional doxycycline (those randomized to PEP arm), d) difficulty
using the smartphone app, or any other study-related concerns. Reimbursement will not be provided for
unscheduled visits. All interim contacts and visits will be documented in participants' study records and on
applicable CRFs.

6.3 Instructions for evaluations

6.3.1 Smart phone application: A smartphone application (app), Blackbook, (developed by AptMobility in
conjuction with UCSF researchers) currently in use to track sexual behavior and PrEP use in young
MSM is being adapted for use in this study to measure daily sexual risk practices (both study arms) and
doxycycline PEP administration (intervention arm only). Study staff will assist with obtaining smartphone
through local available resources, such as Lifeline Assist which make cell phone available to qualifying
lower income individuals.
(https://www.truconnect.com/lifeline/freephone/signup) Participant will be given a code to permit
download from an online resource and guidance on how to use the application.

6.3.2 Review of current medications: All participants will have current medications reviewed at screening for
possible drug interaction with doxycycline. At subsequent quarterly visits, participants randomized to
 20

doxycycline PEP will have current medications be reviewed with participant and any new medications
will be assessed for possible drug interactions.
6.3.3 STI symptom screen. Participants will be asked about current STI symptoms including dysuria, rash,
throat pain, rectal pain/discharge, and genital lesions
6.3.4 General symptom assessement. At each visit, participants in both study arms will be asked about possible
doxycycline side effects, including esophagitis, rash, gastrointestinal symptoms, photosensitivity,
headache, and changes in vision (including blurred, double vision, vision loss). Control arm participants
will be asked about these symptoms to establish a comparator for non-specific symptoms which can
occur in the absence of doxycycline.
6.3.5 Antibiotic and STI history: History of any non-study provided antibiotics taken in the last 3 months and
any STI diagnosis in preceding 3 months. At study entry, STI history for preceding 12 months will be
recorded as well as obtained through chart review.
6.3.6 Meningococal vaccination history: At study entry and at month 12, documention of receipt of any prior
meningococcal vaccination and the type (if known), using a checklist that will include
- Serogroup A only (includes MenAfriVa, PsA-TT)
- Serogroup B only (includes Trumenba, Bexsero)
- Serogroup C only (includes Meningitec, NeisVac C)
- Quadrivalent A/C/Y/W (includes Menomune, Menveo,Nimirix, Menactra)
- Or other formulation
Type of vaccination may be confirmed by chart review if records available.
6.3.7 Sexual behavior questionnaire: Participants will be asked to complete a sexual behavior questionnaire
recording number and type of sexual contacts in the past 3 months at enrollment and between visits
6.3.8 Pill count and doxycycline adherence assessment. Participants assigned to doxycycline PEP will be
asked to bring their doxycycline bottles in at each visit for a pill count. They will be asked what proportion
of sex acts were covered by PEP in the past month and past 3 months, an estimate of overall adherence
(all the time, some of time, half the time, rarely, never), and how many total doses of doxycycline were
taken in the past 3 months
6.3.9 Doxycycline PEP acceptability survey. At months 6 and 12, participants assigned to doxycycline PEP will
be given a brief self-administered questionnaire evaluating acceptability of PEP
6.3.10 Risk reduction counseling and offer to provide of condoms/lubricant At each visit, staff will counsel all
men about risk reduction and HIV (if not living with HIV) and STI prevention, and the importance of
adherence to ART and PrEP.
6.3.11 STI testing.
GC/CT: First void urine and pharyngeal and rectal swabs for NAAT testing. Swabs may be self collected
or collected by study staff. GC & CT testing should be conducted at least 14 days after treatment to avoid
false positive nucleic acid results

RPR: Blood collected for non treponemal assay (RPR or VDRL) with titer if positive. Treponemal assay
results should be recorded if RPR or VDRL are positive. Syphilis testing will be repeated quarterly after
last treatment for syphilis to determine if titers fall or if not, for clinical evaluation of treatment failure or
reinfection. RPR should be conducted at least 30 days after last syphilis treatment and must be
interpreted in the context of prior test results and current symptoms and exposure history. The need for
treatment will be based on the clinical assessment of the site investigator based on the serological data
and clinical findings. All syphilis endpoints will determined by the Endpoint Adjucation committee (see
10.3)

Note: If GC, CT or syphilis testing have been conducted within 30 days prior to study visit and these
results are available, these results may be used for the quarterly STI assessment.

6.3.12 Nasopharyngeal swab for Staph Aureus: nares and oropharynx will be swabbed and cutured for S.
Aureus, with antibiotic resistance testing if S. aureus growth present
 21

6.3.13 Nasal swab for Neisseria species. At baseline, all participants will undergo nares swab for Neisseria
species and TCN-resistance testing if present. Those whose baseline swab show Neisseria spp growth
will have repeat nares swab at month 12.
6.3.14 Stool specimen: Stool samples from the first 50 MSM living with HIV and 50 HIV- MSM on PrEP who
consent to collection will be obtained at baseline, 6 and 12 months for metagenomic resistome studies
using 16sRNA amplification of TCN resistance genes.
6.3.15 Rectal Swab: Rectal swabs will be collected and frozen from all participants who consent for future
microbiome analysis. This may be self collected or collected by study staff.
6.3.16 Hair collection: Hair will be collected from all participants who consent to collection and who meet
eligibility for hair collection (occipital hair length > 0.5 cm and absence of bleaching in area to be collected)
See manual of procedures for details on hair collection
6.3.17 HIV testing and RNA data: At each visit for PLWH, HIV RNA available from clinical visits in the preceding
3 months will be recorded if available and current ART regimen and status (taking vs. not taking) will be
recorded. At each visit for those on PREP, PREP status will be assessed (taking vs. not taking) and
recent HIV test results from clinical care in the preceding 3 months, if available.
6.3.18 Hematologic and liver function tests: Complete blood count (CBC) (white count, hemoglobin, platelets)
and liver function testing (AST, ALT, total bilirubin and alkaline phosphatase) will be drawn at months 3
and 9 in those on doxycycline PEP. Testing done through standard of care within 30 days of the study
visit may also be used if available.
6.3.19 Reimbursement: Participants will be reimbursed at the enrollment visit and at quarterly visits. No
reimbursement will be provided at unscheduled interim visits.

6.4 Management and treatment of GC, CT and syphilis

Individuals with positive GC and CT NAAT results or with serologic and/or clinical presentation indicating a new
syphilis infection will be contacted to return for STI treatment as well as swabs of affected site for resistance
testing or urine collection if the STI is urethral.. Specifically, those with positive GC or CT NAAT results will have
a swab obtained from the involved anatomic site for phenotypic (GC) or a swab or urine obtained (depending on
the site of infection) for molecular resistance testing (CT). For those with positive syphilis serologies, a swab of
mucosal lesions will be obtained, if present, and if no lesions are present, a buccal mucosal swab will be obtained
for T. pallidum DNA amplification which if positive, will be used for resistance testing. For those who have a
presumptive STI at a study visit given characteristic clinical presentation (such as chanre, characteristic rash,
symptomatic urethritis or procititis) such that the treating clinician will provide empiric STI treatmen while awaiting
confirmatory testing, the swab and/or urine collection for resistance testing should be obtained at that time and
prior to treatment, in addition to standard diagnostic testing.

All participants with positive STI test results will be treated according to CDC guidelines. The CDC STI treatment
guidelines will be followed for the treatment of STIs diagnosed by laboratory tests or syndromically (MMWR
Recomm Rep. 2015 Jun 5;64(RR-03):1-137; STD Treatment Guidelines 2015, vol 64). If doxycycline is used for
treatment of an STI for an individual in the PEP arm, doxycycyline PEP will be held during treatment and resumed
when treatment completed.

6.5 Testing for resistence in setting of STI diagnoses on study

6.5.1 Testing for tetracycline resistance among N. gonorrheae

Positive GC cultures for all participants will be sent to either the University of Washington Neisseria Reference
laboratory for resistance testing led by our co-investigators, Dr Olusegun Soge at UW/PHSKC STI program or
the SFDPH Public Health Laboratory by Dr. Godfred Masinde. Both laboratories participate in the Strengthening
the US Response to Resistant Gonorrhea (SURRG) program. GC isolates collected in Seattle and frozen isolates
from San Francisco will be shipped to Dr. Soge on a monthly basis for agar dilution antimicrobial susceptibility
to a panel of antimicrobials including tetracycline, as part of the Antimicrobial Resistance Lab Network (ARLN)
 22

supported by CDC. In addition, Dr. Soge’s laboratory will determine whether tetracycline MICs increases among
the subset with repeat GC infections during follow-up.

6.5.2 Molecular testing for tetracycline resistance genes in C. trachomatis and T. pallidum

DNA from positive NAAT tests for C. trachomatis and from oral mucosal swabs from those with newly diagnosed
syphilis will be evaluated using established CRISPR/Cas9 techniques to screen against a comprehensive library
of antibiotic resistance mutations in order to establish rapid throughput methodology for the multicenter study.
Tetracycline resistance will be assessed using FLASH (Fast Length Adjustment of Short reads) targeted
sequencing of antimicrobial resistance genes that will then be compared to an established antibiotic resistance
database as well as evaluated for known tetracycline mutations that have been documented in the target STIs
or closely related organisms56. Molecular resistance testing will conducted at the San Francisco Chan-
Zuckerberg Biohub under the supervision of collaborating investigators Drs. Langlier and Crawford, who have
extensive expertise in molecular diagnosis of mutations associated with antibiotic resistance. Any TCN-
resistance mutations in C. trachomatis or T. pallidum will be investigated further, as clinically relevant tetracycline
resistance has not been reported to date.

7.0 ADVERSE EVENTS (AE) AND STUDY MONITORING

7.1 Adverse Event Collection Requirements

All AEs must be recorded on eCRFs if any of the following criteria have been met:
• All grade 2 and higher hematologic and hepatic laboratory abnormalities that are attributed to
doxycycline in the opinion of the site investigator
• All AEs meeting SAE definition

Serious Adverse Events (SAEs)

An SAE is defined as any untoward medical occurrence that:
• Results in death
• Is life-threatening
• Requires inpatient hospitalization or prolongation of existing hospitalization
• Results in persistent or significant disability/incapacity
• Is an important medical event that may not be immediately life-threatening or result in death or
hospitalization but may jeopardize the patient or may require intervention to prevent one of the
other outcomes listed in the definition above)

All AEs that are recorded must have their severity graded. To grade AEs, sites should refer to the Division of
AIDS Table for Grading the Severity of Adult and Pediatric Adverse Events (DAIDS AE Grading Table),
corrected Version 2.1, July 2017, which can be found on the DAIDS RSC website at https://rsc.tech-
res.com/docs/default-source/safety/division-of-aids-(daids)-table-for-grading-the-severity-of-adult-and-
pediatric-adverse-events-corrected-v-2-1.pdf

7.2 AE and SAE attribution to doxycycline

In those randomized to receive doxycycline PEP, all AE’s and SAE’s should have attribution recorded as
doxycycline-related or not doxycycline-related, in the judgment of the site investigator.

7.3 Study Monitoring

 23

7.3.1 Study Team Monitoring

The study team will monitor the conduct of the study through monthly summary reports of arms of accrual, and
baseline characteristics and quarterly reports of data pooled over treatment arms of data completeness,
specimen collection, and AEs. The study will review individual participant-level safety data frequently to assess
the relation of all reported AEs to study treatment. On a monthly basis, the study team will review by-arm
summaries of premature study discontinuations and premature study treatment discontinuations (and reasons)
and AEs.

7.3.2 Independent Monitor

Study conduct will be monitored by an independent monitor. Monitors will visit participating clinical research
sites to review the individual participant records, including consent forms, eCRFs, supporting data, laboratory
specimen records, and medical records (physicians’ progress notes, nurses’ notes, individuals’ hospital charts),
to ensure protection of study participants, compliance with the protocol, and accuracy and completeness of
records. The monitors also will inspect sites’ regulatory files to ensure that regulatory requirements are being
followed and sites’ pharmacies to review product storage and management.

7.3.3 DSMB
An independent data safety and monitoring board (DSMB) will be convened for this study with expertise in STIs,
PrEP, and HIV care and a biostatistician. The purpose of the DSMB is to monitor the study for operational futility,
social harms, and efficacy. The DSMB will evaluate the progress of the project, including periodic assessments
of accrual, retention, safety, performance and variation of the project sites, and other factors that can affect
project implementation. The DSMB will review the study after 1/3 and 2/3 follow-up time with pre-specified
stopping rules for efficacy and futility in terms of the efficacy of doxycycline PEP in reducing the incidence of
gonorrhea, chlamydia, and syphilis overall, and the ability of the study to meet its objectives. The DSMB will
review STI endpoints which have been adjudicated by the STI Endpoints Committee. The DMSB will consider
factors external to the project when relevant information becomes available, such as policy changes or scientific
developments that may have an impact on project implementation, safety, and integration of STI PEP in the STI
and HIV care clinics.

The DSMB will conduct reviews every six month and convene by teleconference. Open reports containing
accrual and retention rates, participant characteristics, serious adverse events, and social, will be sent to the
protocol team and DSMB members the week prior to the DSMB meeting. Only the DSMB members and the
unblinded biostatistician will receive password-protected closed reports of STI endpoints by randomization arm.

8.0 CLINICAL MANAGEMENT ISSUES

8.1 Toxicity

Only toxicities related to study medications provided through the study will be considered in the toxicity
management section.

Grade 1 or 2
Participants who develop Grade 1 or 2 toxicity (per DAIDS toxicity table) felt to be related to
doxycycyline may continue study treatment at the discretion of the site investigator with close follow-up.
If a participant chooses to discontinue study treatment, the site should notify the study protocol team
within 7 days.These participants will remain on study, off study treatment and have all evaluations
performed per the SOE.

Grade 3
 24

• Participants who develop a Grade 3 toxicity thought by the site investigator to be related to study
drug should have study drug withheld and the site should consult with the core protocol team. The
participant should be reevaluated weekly until the AE returns to Grade ≤2, at which time study drug
may be reintroduced at the discretion of the site investigator in consultation with the protocol team.
• Participants experiencing Grade 3 toxicity requiring permanent discontinuation of study drug should
be followed weekly until resolution of the toxicity. Participants will have premature study treatment
discontinuation evaluations performed as noted on the SOE. These participants will remain on
study, off study treatment and have all evaluations performed per the SOE.

Grade 4
• Participants with Grade 4 asymptomatic laboratory abnormalities may continue study drug if the site
investigator has compelling evidence that the toxicity is NOT related to study drug.
• Participants who develop a Grade 4 symptomatic toxicity will have study drug permanently
discontinued and the site should notify study team within 72 hours.
• Participants experiencing Grade 4 toxicity requiring permanent discontinuation of study drug should
be followed weekly until resolution of the AE or return to baseline. These participants will remain on
study, off study treatment and have all evaluations performed per the SOE.

8.2 Specific Management of Toxicities Related to Study-Provided Drugs

Possible intracranial hypertension (IH)

Participants taking doxycycline who report new or worsening headaches, visual changes or vision loss
should have doxycycline temporarily discontinued and an evaluated for IH, including a fundoscopic exam
to look for papilledema. If IH is rule out or an alternate etiology identified, doxycycline may be restarted,
at the discretion of the site investigator.

Skin erythema
Increased photosensivity is a known possible side effect of doxycycline. Doxycycline should be
discontinued if skin erythema develops and may reinstituted when resolved at the discretion of the site
investigation. Any serious skin reaction should lead to permanent discontinuation of doxycycline .

Fixed drug eruption

Suspected fixed drug eruption should be evaluated for possible etiologies – if a doxycycline fixed drug
eruption is suspected, doxycycline should be stopped.

Allergic reactions
Doxycycline should be discontinued permanently if an allergic reaction is suspected. These participants
will remain on study, off study treatment and have all evaluations performed per the SOE.

9.0 CRITERIA FOR DISCONTINUATION

9.1 Premature Study Treatment Discontinuation

• Requirement for prohibited concomitant medications or other contraindication to doxycycline

• Occurrence of an adverse event requiring discontinuation of doxycycline
• Request by participant to terminate study treatment
• Clinical reasons believed life threatening by the physician, even if not addressed in the toxicity
section of the protocol
• Requirement for chronic tetracycline use ( >14 days)
 25

Participants who stop doxycycline PEP should be continued on study, off PEP with continued
evaluations as per the SOE. The reason for doxycycline discontinuation should be recorded.

9.2 Premature Study Discontinuation

• Request by the participant to withdraw

• Request of the primary care provider if she or he thinks the study is no longer in the best interest of
the participant
• Participant judged by the investigator to be at significant risk of failing to comply with the provisions
of the protocol as to cause harm to self or seriously interfere with the validity of the study results
• At the discretion of the IRB/EC, NIAID, Office for Human Research Protections (OHRP), other
government agencies as part of their duties, investigator, or industry supporter

10.0 STATISTICAL CONSIDERATIONS

10.1 Outcome measures

10.1.1 Primary outcome: Combined incidence of GC, CT, or early syphilis infection by by laboratory-
based diagnosis (e.g., positive GC or CT based on NAAT, syphilis based on four-fold increase in non-
treponemal titers or positive darkfield or fluorescent treponemal antibody on exudate from a lesion).
Incident STI diagnoses will be ascertained through the study through periodic testing, interim visits for
STI symptoms, as well as STI surveillance from the San Francisco and King County Departments of
Health and by medical records provided for participants who receive their care from other clinical sites.

10.1.2 Secondary outcomes:

• Safety of doxycycline PEP, measured by evaluation of all Grade 3 and 4 AEs attributed to study
medication.
• Tolerability, measured using a brief quarterly questionnaire administered to both intervention and
control groups evaluating side effects commonly associated with doxycycline as well as recording
any discontinuations of doxycycline by participants and their reasons.
• Acceptability, assessed through a brief questionnaire for all PEP recipients administered at month 6
and 12 and in-depth structured qualitative interviews for a subset.
• Doxycycline coverage of sexual contacts in the prior 3 months,ascertained by sexual behavior and
doxycycline use from the web-based app, augmented by quarterly adherence questionnaires and pill
counts.
• Longitudinal exposure to doxcycyline will be assessed using hair collection, with a semiquantitive
evaluation in the control arm (doxycycline present vs. absent) and in the PEP arm ( doxycycline
concentration high vs. low).
• Adherence to HIV medications among HIV-infected MSM, assessed by chart review of HIV RNA
levels conducted as standard clinical care.
• Residual hair samples of HIV-uninfected MSM on PrEP will be archived for future tenofovir testing to
measure adherence to Truvada PrEP.

10.2 Statistical power and analysis

Primary endpoint: We selected a sample size for each cohort (HIV-infected MSM and HIV-uninfected MSM on
PrEP) to achieve 80% power with 0.05 two-sided Type 1 error, for the endpoint of any lab-detected incident early
syphilis, GC and/or CT infection, based on quarterly assessments for each person, 10% annual loss to follow-
up and an intra-class correlation of 0.2, reflecting the high prevalence of re-infection that has been observed in
these populations. With these assumptions, a cohort of 390 of HIV-infected and 390 of HIV uninfected MSM on
PrEP (130 in the control and 260 in the intervention for each cohort) will provide 80% power to detect a decrease
in quarterly STI prevalence from 10% to 5%, which corresponds to an annual reduction in combined STI
 26

incidence from 34% to 19%. With allowance for 10% lost to follow-up, the sample size for each cohort will be
390 participants, for a total sample size of 780. Based on 2017 STI incidence data from San Francisco and
Seattle PrEP cohorts for both GC and CT, we expect in the control arm in each quarter approximately 5% of
participants to have GC, 5% CT and 2% early syphilis, acknowledging some will be diagnosed with more than
one infection. Based on the higher susceptibility of GC to TCN in the US vs France (75% vs < 50%13, 22) and the
70% reduction of CT and syphilis with PEP in IPERGAY,1 we anticipate effectiveness of doxycycline PEP to be
35% for GC, 65% for CT and 65% for syphilis. Given these assumptions, with a 2:1 randomization we expect to
see 61 vs 47 GC infections, 47 vs 33 CT infections and 19 vs 13 syphilis infections in the doxycycline PEP and
control arms respectively, some of which will occur in the same individuals. Sample sizes were computed using
nQuery for repeated measures for two proportions. The analysis will compare time-averaged proportion of
infections in each quarter between arms, using repeated measure methods.

Secondary endpoints include evaluation of PEP effectiveness on incidence of each specific STI and on time to
first STI, as well as by site of STI infection (oral, rectal, urethral). Impact of risk practices and sexual activity on
STI incidence will be compared between arms. Survey data will be analyzed to understand factors impacting
acceptability of doxycycline PEP, including demographics factors, socioeconomic status, substance use, history
of STI, HIV infection status, and experience with PEP. The qualitative interviews will be coded using inductive
(i.e. codes arise from careful reading of the text) and deductive (i.e. codes arise a priori from topics covered by
the interview guide) approaches, which when employed together have proven useful in health services research.
Interviews will be coded by at least two research team members who will practice on a selection of transcripts
until >90% agreement is reached. Dedoose, a web-based program, will be used to facilitate indexing, coding,
and searching. The qualitative team will meet regularly to group codes into categories and develop major and
minor themes.

For resistance we will assess the proportion with tetracycline resistance in visits with N. gonorrheae and S.
aureus. Exploratory endpoints include incidence of tetracycline resistance in C. trachomatis and T. pallidum
using molecular techniques (e.g., CRISPR and CAS-9), for which the molecular technologies are new and the
clinical significance of resistance mutations is less clear. Stool samples from 50 MSM living with HIV and 50 HIV-
uninfected MSM on PrEP will be obtained at baseline, 6 and 12 months for metagenomic evaluation targeting
TCN genes. Overall TCN resistance gene abundance (expressed as dpM/patient) and the number of distinct
TCN resistance genes present per participant will be assessed at baseline and evaluated for change over time
during doxycycline use. Rectal swabs will be collected and stored for future evaluation of shifts in the fecal
microbiome, decrease in flora diversity, and quantification of TCN-resistant abundance and diversity

10.3 Endpoint adjudication

All STI endpoints will be reviewed by a blinded, independent Endpoint Adjudication Committee, following CDC
guidelines for the diagnosis of STIs.57 Incidence of individual STIs and tetracycline resistance will also be
evaluated.

12.0 PARTICIPANTS

12.1 Institutional Review Board (IRB) Review

The study protocol, site-specific informed consent forms, participant education and recruitment materials, and
other requested documents—including any subsequent modifications—will be reviewed and approved by the
UCSF IRB, as the single IRB of record responsible for oversight of research conducted at the study sites.
Subsequent to initial review and approval, the UCSF IRB will review the study at least annually.

12.2 Informed Consent

 27

A signed consent form will be obtained from the participant .The consent form will describe the purpose of the
study, the procedures to be followed, and the risks and benefits of participation. A copy of the consent form will
be given to the participant and this fact will be documented in the participant’s record.

12.3 Study records

Each study site will establish a standard operating procedure for confidentiality protection. Each site will ensure
that study records including administrative documentation and regulatory documentation as well as
documentation related to each participant enrolled in the study, including informed consent forms, locator forms,
case report forms, notations of all contacts with the participant, and all other source documents are stored in a
secure manner.

12.4 Confidentiality

Participants’ study information will not be released without their written permission, except as necessary for
oversight by:
- The Protocol co-Chairs or designees
- Study funders
- University of California, San Francisco IRB
- University of Washington IRB

All laboratory specimens, evaluation forms, reports, and other records that leave the site will be identified by
coded number only to maintain participant confidentiality. The exceptionare STI testing results which are subject
to local and state reporting which is names-based. Local public health may contact paricipants diagnosed with
STIs for the purpose of surveillance and partner notification. Participants will be informed prior to STI testing
that results are reportable and may lead to contact by local public health if results are positive for infeciotn. In
addition, GC culture specimens will be evaluated for drug resistance as part of an existing GC program evaluating
clinical isolates may contain patient identifiers such as MRN, name or date of visit, according to local laboratory
requirements and practice.

All records will be kept locked. All computer entry and networking programs will be done with coded numbers
only. Clinical information will not be released without written permission of the participant, except as necessary
for monitoring by the IRB/EC, NIAID, OHRP, other local, US, and international regulatory entities as part of their
duties, or the industry supporters or designees.

13.0 BIOHAZARD CONTAINMENT

As the transmission of HIV and other blood-borne pathogens can occur through contact with contaminated
needles, blood, and blood products, appropriate blood and secretion precautions will be employed by all
personnel in the drawing of blood and shipping and handling of all specimens for this study, as currently
recommended by the CDC and the National Institutes of Health.

All dangerous goods and materials, including diagnostic specimens and infectious substances, must be
transported using packaging mandated by CFR 42 Part 72. Please refer to instructions detailed in the
International Air Transport Association (IATA) Dangerous Goods Regulations.
 28

References

1. Molina, J.M., I. Charreau, C. Chidiac, G. Pialoux, E. Cua, C. Delaugerre, C. Capitant, D. Rojas-Castro,

J. Fonsart, B. Bercot, C. Bebear, L. Cotte, O. Robineau, F. Raffi, P. Charbonneau, A. Aslan, J. Chas, L.
Niedbalski, B. Spire, L. Sagaon-Teyssier, D. Carette, S.L. Mestre, V. Dore, and L. Meyer, Post-
exposure prophylaxis with doxycycline to prevent sexually transmitted infections in men who have sex
with men: an open-label randomised substudy of the ANRS IPERGAY trial. Lancet Infect Dis, 2017.

2. SFDPH. San Francisco Sexually Transmitted Disease Annual Summary, 2015. 2017; Available from:
https://www.sfdph.org/dph/files/reports/StudiesData/STD/SFSTDAnnlSum2015.pdf.

3. King County Sexually Transmitted Diseases Epidemiology Report (2015). 2015.

4. CDC, Sexually Transmitted Disease Surveillance 2016. 2017.

5. Volk, J.E., J.L. Marcus, T. Phengrasamy, D. Blechinger, D.P. Nguyen, S. Follansbee, and C.B. Hare,
No New HIV Infections With Increasing Use of HIV Preexposure Prophylaxis in a Clinical Practice
Setting. Clin Infect Dis, 2015. 61(10): p. 1601-3.

6. Montano, M.A., J.C. Dombroski, L.A. Barbee, M.R. Golden, and C.M. Khosropour. Changes in sexual
behavior and STI diagnoses among MSM using PrEP in Seattle, WA (Abstract #979). in Conference on
Retroviruses and Opportunistic Infections (CROI). 2018. Boston, MA.

7. Oliver, S.E., M. Aubin, L. Atwell, J. Matthias, A. Cope, V. Mobley, A. Goode, S. Minnerly, J. Stoltey,
H.M. Bauer, R.R. Hennessy, D. DiOrio, R.N. Fanfair, T.A. Peterman, and L. Markowitz, Ocular Syphilis
- Eight Jurisdictions, United States, 2014-2015. MMWR Morb Mortal Wkly Rep, 2016. 65(43): p. 1185-
1188.

8. Bowen, V., J. Su, E. Torrone, S. Kidd, and H. Weinstock, Increase in incidence of congenital syphilis -
United States, 2012-2014. MMWR Morb Mortal Wkly Rep, 2015. 64(44): p. 1241-5.

9. Cunningham, S.D., G. Olthoff, P. Burnett, A.M. Rompalo, and J.M. Ellen, Evidence of heterosexual
bridging among syphilis‐positive men who have sex with men. Sexually Transmitted Infections, 2006.
82(6): p. 444-445.

10. Wi, T., M.M. Lahra, F. Ndowa, M. Bala, J.-A.R. Dillon, P. Ramon-Pardo, S.R. Eremin, G. Bolan, and M.
Unemo, Antimicrobial resistance in Neisseria gonorrhoeae: Global surveillance and a call for
international collaborative action. PLOS Medicine, 2017. 14(7): p. e1002344.

11. Gianecini, R., C. Oviedo, G. Stafforini, and P. Galarza, Neisseria gonorrhoeae Resistant to Ceftriaxone
and Cefixime, Argentina. Emerging Infectious Diseases, 2016. 22(6): p. 1139-1141.

12. Camara, J., J. Serra, J. Ayats, T. Bastida, D. Carnicer-Pont, A. Andreu, and C. Ardanuy, Molecular
characterization of two high-level ceftriaxone-resistant Neisseria gonorrhoeae isolates detected in
Catalonia, Spain. J Antimicrob Chemother, 2012. 67(8): p. 1858-60.

13. Kirkcaldy, R.D., A. Harvey, J.R. Papp, C. Del Rio, O.O. Soge, K.K. Holmes, E.W. Hook, 3rd, G. Kubin,
S. Riedel, J. Zenilman, K. Pettus, T. Sanders, S. Sharpe, and E. Torrone, Neisseria gonorrhoeae
Antimicrobial Susceptibility Surveillance - The Gonococcal Isolate Surveillance Project, 27 Sites, United
States, 2014. MMWR Surveill Summ, 2016. 65(7): p. 1-19.
 29

14. BBC. 'World's worst' super-gonorrhoea man cured. 2018 4/26/2018]; Available from:
http://www.bbc.com/news/health-43840505.

15. Lahra, M.M., N. Ryder, and D.M. Whiley, A new multidrug-resistant strain of Neisseria gonorrhoeae in
Australia. N Engl J Med, 2014. 371(19): p. 1850-1.

16. Cohen, M.S., I.F. Hoffman, R.A. Royce, P. Kazembe, J.R. Dyer, C.C. Daly, D. Zimba, P.L. Vernazza,
M. Maida, S.A. Fiscus, and J.J. Eron, Jr., Reduction of concentration of HIV-1 in semen after treatment
of urethritis: implications for prevention of sexual transmission of HIV-1. AIDSCAP Malawi Research
Group. Lancet, 1997. 349(9069): p. 1868-73.

17. Schacker, T., A.J. Ryncarz, J. Goddard, K. Diem, M. Shaughnessy, and L. Corey, Frequent recovery of
HIV-1 from genital herpes simplex virus lesions in HIV-1-infected men. JAMA, 1998. 280(1): p. 61-6.

18. CDC. Understanding the HIV Care Continuum. 2017; Available from:
https://www.cdc.gov/hiv/pdf/library/factsheets/cdc-hiv-care-continuum.pdf.

19. The Lancet, H., U=U taking off in 2017. Lancet HIV, 2017. 4(11): p. e475.

20. Harrison, W.O., R.R. Hooper, P.J. Wiesner, A.F. Campbell, W.W. Karney, G.H. Reynolds, O.G. Jones,
and K.K. Holmes, A trial of minocycline given after exposure to prevent gonorrhea. N Engl J Med, 1979.
300(19): p. 1074-8.

21. Steen, R., M. Chersich, A. Gerbase, G. Neilsen, A. Wendland, F. Ndowa, E.A. Akl, Y.R. Lo, and S.J. de
Vlas, Periodic presumptive treatment of curable sexually transmitted infections among sex workers: a
systematic review. Aids, 2012. 26(4): p. 437-45.

22. La Ruche, G., A. Goubard, B. Bercot, E. Cambau, C. Semaille, and P. Sednaoui, Gonococcal infections
and emergence of gonococcal decreased susceptibility to cephalosporins in France, 2001 to 2012.
Euro Surveill, 2014. 19(34).

23. Molina, J.M., I. Charreau, C. Chidiac, G. Pialoux, E. Cua, C. Delaugerre, C. Capitant, D. Rojas-Castro,
J. Fonsart, B. Bercot, C. Bebear, L. Cotte, O. Robineau, F. Raffi, P. Charbonneau, A. Aslan, J. Chas, L.
Niedbalski, B. Spire, L. Sagaon-Teyssier, D. Carette, S.L. Mestre, V. Dore, and L. Meyer. Supplement
to: Molina JM, Charreau I, Chidiac C, et al. Post-exposure prophylaxis with doxycycline to prevent
sexually transmitted infections in men who have sex with men: an open-label randomised substudy of
the ANRS IPERGAY trial. Lancet Infect Dis 2017. 2017; Available from:
https://www.sciencedirect.com/science/article/pii/S1473309917307259?via%3Dihub#sec1.

24. Bolan, R.K., M.R. Beymer, R.E. Weiss, R.P. Flynn, A.A. Leibowitz, and J.D. Klausner, Doxycycline
prophylaxis to reduce incident syphilis among HIV-infected men who have sex with men who continue
to engage in high-risk sex: a randomized, controlled pilot study. Sex Transm Dis, 2015. 42(2): p. 98-
103.

25. Gaillard, T., M. Madamet, and B. Pradines, Tetracyclines in malaria. Malar J, 2015. 14: p. 445.

26. van Zuuren, E.J. and Z. Fedorowicz, Interventions for rosacea: abridged updated Cochrane systematic
review including GRADE assessments. Br J Dermatol, 2015. 173(3): p. 651-62.

27. Bienenfeld, A., A.R. Nagler, and S.J. Orlow, Oral Antibacterial Therapy for Acne Vulgaris: An Evidence-
Based Review. Am J Clin Dermatol, 2017. 18(4): p. 469-490.
 30

28. Sloan, B. and N. Scheinfeld, The use and safety of doxycycline hyclate and other second-generation
tetracyclines. Expert Opin Drug Saf, 2008. 7(5): p. 571-7.

29. Turner, R.B., C.B. Smith, J.L. Martello, and D. Slain, Role of doxycycline in Clostridium difficile infection
acquisition. Ann Pharmacother, 2014. 48(6): p. 772-6.

30. Tariq, R., J. Cho, S. Kapoor, R. Orenstein, S. Singh, D.S. Pardi, and S. Khanna, Low Risk of Primary
Clostridium difficile Infection With Tetracyclines: A Systematic Review and Metaanalysis. Clin Infect
Dis, 2018. 66(4): p. 514-522.

31. Doernberg, S.B., L.G. Winston, D.H. Deck, and H.F. Chambers, Does doxycycline protect against
development of Clostridium difficile infection? Clin Infect Dis, 2012. 55(5): p. 615-20.

32. Khanna, S., Do tetracyclines have the potential to reduce the risk of Clostridium difficile infection?
Expert Review of Anti-infective Therapy, 2018: p. 1-3.

33. Magnuson, H.J., H. Eagle, and R. Fleischman, The minimal infectious inoculum of Spirochaeta pallida
(Nichols strain) and a consideration of its rate of multiplication in vivo. Am J Syph Gonorrhea Vener Dis,
1948. 32(1): p. 1-18.

34. Cumberland, M.C. and T.B. Turner, The rate of multiplication of Treponema pallidum in normal and
immune rabbits. Am J Syph Gonorrhea Vener Dis, 1949. 33(3): p. 201-12.

35. Lafond, R.E. and S.A. Lukehart, Biological basis for syphilis. Clin Microbiol Rev, 2006. 19(1): p. 29-49.

36. Nadelman, R.B., J. Nowakowski, D. Fish, R.C. Falco, K. Freeman, D. McKenna, P. Welch, R. Marcus,
M.E. Aguero-Rosenfeld, D.T. Dennis, and G.P. Wormser, Prophylaxis with single-dose doxycycline for
the prevention of Lyme disease after an Ixodes scapularis tick bite. N Engl J Med, 2001. 345(2): p. 79-
84.

37. Levett, P.N., Leptospirosis. Clin Microbiol Rev, 2001. 14(2): p. 296-326.

38. Schneider, M.C., J. Velasco-Hernandez, K.D. Min, D.G. Leonel, D. Baca-Carrasco, M.E. Gompper, R.
Hartskeerl, and C. Munoz-Zanzi, The Use of Chemoprophylaxis after Floods to Reduce the Occurrence
and Impact of Leptospirosis Outbreaks. Int J Environ Res Public Health, 2017. 14(6).

39. Burkot, T.R., J. Piesman, and R.A. Wirtz, Quantitation of the Borrelia burgdorferi outer surface protein A
in Ixodes scapularis: fluctuations during the tick life cycle, doubling times, and loss while feeding. J
Infect Dis, 1994. 170(4): p. 883-9.

40. Wanninger, S., M. Donati, A. Di Francesco, M. Hässig, K. Hoffmann, H.M.B. Seth-Smith, H. Marti, and
N. Borel, Selective Pressure Promotes Tetracycline Resistance of Chlamydia Suis in Fattening Pigs.
PLOS ONE, 2016. 11(11): p. e0166917.

41. Dugan, J., D.D. Rockey, L. Jones, and A.A. Andersen, Tetracycline resistance in Chlamydia suis
mediated by genomic islands inserted into the chlamydial inv-like gene. Antimicrob Agents Chemother,
2004. 48(10): p. 3989-95.

42. Mirajkar, N.S., P.R. Davies, and C.J. Gebhart, Antimicrobial Susceptibility Patterns of Brachyspira
Species Isolated from Swine Herds in the United States. J Clin Microbiol, 2016. 54(8): p. 2109-19.
 31

43. Centurion-Lara, A., C. Castro, W.C. van Voorhis, and S.A. Lukehart, Two 16S-23S ribosomal DNA
intergenic regions in different Treponema pallidum subspecies contain tRNA genes. FEMS Microbiol
Lett, 1996. 143(2-3): p. 235-40.

44. Mitchell, S.J., J. Engelman, C.K. Kent, S.A. Lukehart, C. Godornes, and J.D. Klausner, Azithromycin-
resistant syphilis infection: San Francisco, California, 2000-2004. Clin Infect Dis, 2006. 42(3): p. 337-
45.

45. Stamm, L.V., Global challenge of antibiotic-resistant Treponema pallidum. Antimicrob Agents
Chemother, 2010. 54(2): p. 583-9.

46. Lucet, J.-C. and B. Regnier, Screening and Decolonization: Does Methicillin-Susceptible
Staphylococcus aureus Hold Lessons for Methicillin-Resistant S. aureus? Clinical Infectious Diseases,
2010. 51(5): p. 585-590.

47. Stevens, D.L., A.L. Bisno, H.F. Chambers, E.P. Dellinger, E.J. Goldstein, S.L. Gorbach, J.V.
Hirschmann, S.L. Kaplan, J.G. Montoya, J.C. Wade, and A. Infectious Diseases Society of, Practice
guidelines for the diagnosis and management of skin and soft tissue infections: 2014 update by the
Infectious Diseases Society of America. Clin Infect Dis, 2014. 59(2): p. e10-52.

48. Jakobsson, H.E., C. Jernberg, A.F. Andersson, M. Sjölund-Karlsson, J.K. Jansson, and L. Engstrand,
Short-Term Antibiotic Treatment Has Differing Long-Term Impacts on the Human Throat and Gut
Microbiome. PLOS ONE, 2010. 5(3): p. e9836.

49. Shanahan, F., The colonic microbiota in health and disease. Curr Opin Gastroenterol, 2013. 29(1): p.
49-54.

50. Blaser, M.J., The Past and Future Biology of the Human Microbiome in an Age of Extinctions. Cell,
2018. 172(6): p. 1173-1177.

51. Petousis-Harris, H., J. Paynter, J. Morgan, P. Saxton, B. McArdle, F. Goodyear-Smith, and S. Black,
Effectiveness of a group B outer membrane vesicle meningococcal vaccine against gonorrhoea in New
Zealand: a retrospective case-control study. Lancet, 2017. 390(10102): p. 1603-1610.

52. ACIP. Recommended Immunization Schedule for Adults Aged 19 Years or Older, United States, 2018.
2018; Available from: https://www.cdc.gov/vaccines/schedules/hcp/imz/adult.html#f10.

53. Angelakis, E., N. Armstrong, C. Nappez, M. Richez, E. Chabriere, and D. Raoult, Doxycycline assay
hair samples for testing long-term compliance treatment. Journal of Infection, 2015. 71(5): p. 511-517.

54. Lexicomp, Doxycycline (Lexi-Drugs) 2019.

55. Mayne. Doxycycline Prescribing information 1/5/2019]; Available from:

https://dailymed.nlm.nih.gov/dailymed/getFile.cfm?setid=54ce5887-5e72-4d7a-a821-
936dd3ffa68c&type=pdf&name=54ce5887-5e72-4d7a-a821-936dd3ffa68c

56. Jia, B., A.R. Raphenya, B. Alcock, N. Waglechner, P. Guo, K.K. Tsang, B.A. Lago, B.M. Dave, S.
Pereira, A.N. Sharma, S. Doshi, M. Courtot, R. Lo, L.E. Williams, J.G. Frye, T. Elsayegh, D. Sardar,
E.L. Westman, A.C. Pawlowski, T.A. Johnson, F.S. Brinkman, G.D. Wright, and A.G. McArthur, CARD
2017: expansion and model-centric curation of the comprehensive antibiotic resistance database.
Nucleic Acids Res, 2017. 45(D1): p. D566-D573.
 32

57. Workowski, K.A., G.A. Bolan, C. Centers for Disease, and Prevention, Sexually transmitted diseases
treatment guidelines, 2015. MMWR Recomm Rep, 2015. 64(RR-03): p. 1-137.
 33

PROTOCOL

DoxyPEP: Evaluation of doxycycline post-exposure prophylaxis to reduce sexually transmitted infections in

men who have sex with men and transgender women either on PrEP or living with HIV

Version 9.0

May 27, 2022

 34

TABLE OF CONTENTS

STUDY TEAM ................................................................................................................................................... 4

GLOSSARY....................................................................................................................................................... 5
SCHEMA ........................................................................................................................................................... 6
1 BACKGROUND AND SIGNIFICANCE....................................................................................................... 8
1.1 Background ........................................................................................................................................ 8
1.2 Rationale .......................................................................................................................................... 10
2 STUDY DESIGN ...................................................................................................................................... 13
3 OBJECTIVES .......................................................................................................................................... 14
3.1 Primary Objectives ........................................................................................................................... 14
3.2 Secondary Objectives ...................................................................................................................... 14
3.3 Exploratory objectives ...................................................................................................................... 14
4 SELECTION AND ENROLLMENT OF PARTICIPANTS .......................................................................... 14
4.1 Inclusion criteria ............................................................................................................................... 15
4.2 Exclusion criteria .............................................................................................................................. 15
4.3 Recruitment...................................................................................................................................... 15
4.4 Co-enrollment guidelines. ................................................................................................................. 16
5 STUDY TREATMENT .............................................................................................................................. 16
5.1 Study product ................................................................................................................................... 16
5.2 Safety of doxycycline ....................................................................................................................... 16
5.3 Doxycycline dispensing and administration. ..................................................................................... 17
5.4 Concomitant medications ................................................................................................................. 17
5.5 HIV pre-exposure prophylaxis (PrEP) and HIV antiretroviral therapy (ART) ..................................... 17
6 CLINICAL AND LABORATORY MONITORING ....................................................................................... 18
6.1 Schedule of Events .......................................................................................................................... 18
6.2 Study visits ....................................................................................................................................... 20
6.3 Instructions for evaluations ............................................................................................................... 20
6.4 Evaluation at time of presumptive or diagnosed GC, CT and syphilis ............................................... 23
6.5 Testing for resistance in setting of STI diagnoses on study .............................................................. 25
6.6 Treatment of STIs ............................................................................................................................ 26
6.7 Qualitative interviews ....................................................................................................................... 26
6.8 Reenrollment of participants enrolled prior to 3/15/2020 ................................................................... 26
7 ADVERSE EVENTS (AE) AND STUDY MONITORING ........................................................................... 26
7.1 Adverse Event Collection Requirements .......................................................................................... 26
7.2 AE and SAE attribution to doxycycline ............................................................................................. 27
7.3 Study Monitoring .............................................................................................................................. 27
8 CLINICAL MANAGEMENT ISSUES ........................................................................................................ 28
8.1 Toxicity ............................................................................................................................................. 28
 35

8.2 Specific Management of Toxicities Related to Study-Provided Drugs............................................... 28

9 CRITERIA FOR DISCONTINUATION ...................................................................................................... 29
9.1 Premature Study Treatment Discontinuation .................................................................................... 29
9.2 Premature Study Discontinuation ..................................................................................................... 29
10 STATISTICAL CONSIDERATIONS ......................................................................................................... 29
10.1 Outcome measures .......................................................................................................................... 29
10.2 Statistical power and analysis .......................................................................................................... 30
10.3 Endpoint adjudication ....................................................................................................................... 31
10.4 Post COVID follow-up ...................................................................................................................... 32
10.5 Analyses post 5/13/2022 closure to enrollment ................................................................................ 32
11 PARTICIPANTS....................................................................................................................................... 32
11.1 Institutional Review Board (IRB) Review .......................................................................................... 32
11.2 Informed Consent............................................................................................................................. 32
11.3 Study records ................................................................................................................................... 32
11.4 Confidentiality .................................................................................................................................. 33
12 BIOHAZARD CONTAINMENT ................................................................................................................. 33
REFERENCES ................................................................................................................................................ 34
 36

STUDY TEAM

Connie Celum, MD, MPH, co-Principal Investigator

Annie Luetkemeyer, MD, co-Principal Investigator
Jared Baeten, MD, PhD
Ruanne Barnabas, MBChB, DPhil
Susan Buchbinder, MD, MPH
Katerina Christopoulos, MD, MPH
Edwin D. Charlebois, MPH, PhD
Stephanie Cohen, MD, MPH
Julie Dombrowski, MD, MPH
Deborah Donnell, PhD
Rob Fredericksen, PhD
Diane Havlir, MD
Chaz Langelier, MD,PhD
Hyman Scott, MD, MPH
Olusegun Soge, MSc, PhD
Vivek Jain, MD, MAS
Emma Bainbridge, MD, MPH
 37

GLOSSARY

AE Adverse event
AMR Antimicrobial resistance
ART Antiretroviral therapy
CT Chlamydia trachomatis
GC Gonorrea
HIV Human Immunodeficiency virus
LGV Lymphogranuloma venereum
MG Mycoplasma genitalium
MSM Men who have sex with men
NAAT Nucleic acid amplification test
NGU Non-gonococcal urethritis
PEP Post-exposture prophylaxis
PLWH People living with HIV
PrEP Pre-exposure prophylaxis
RPR Rapid Plasma Reagin
SAE Serious Adverse Event
STI Sexually transmitted infection
TCN Tetracycline
TGW Transgender women
VDRL Venereral Disease Research Lab
 38

SCHEMA

DoxyPEP: Prospective, randomized, open-label, clinical trial to evaluate the impact of doxycycline post-
exposure prophylaxis on the occurrence of bacterial STIs among men who have sex with men (MSM) and
transgender women (TGW) PrEP users and people living with HIV

Design: Open-label randomized clinical trial of doxycyline PEP to reduce bacterial STIs (N.
gonorrheae [GC], C. trachomatis [CT], and T. pallidum [syphilis]) among MSM and
transgender women (TGW) living with HIV (PLWH) or on HIV PrEP.

Study Population: Sample size of 780, approximately equal numbers of PLWH and participants taking HIV pre-
exposure prophylaxis (PrEP)
Study Sites:
1. HIV/AIDS Clinic (“Ward 86”), Zuckerberg San Francisco General Hospital, UCSF, San
Francisco
2. San Francisco City Clinic, San Francisco Department of Public Health, San Francisco
(municipal STD clinic)
3. Madison HIV/AIDS Clinic, Harborview Medical Center University of Washington, Seattle
4. Public Health-Seattle & King County Sexual Health Clinic at Harborview Medical Center,
Seattle
Primary Study Objectives:
1. Evaluate the effectiveness of doxycycline post-exposure prophylaxis (PEP) to reduce
STI infections in two populations: a) MSM living with HIV and b) MSM/TGW taking HIV
PrEP
2. Investigate the impact of doxycycline PEP on development of culture-based tetracycline
(TCN) resistance in N. gonorrheae and among nasopharyngeal carriers of S. aureus

Secondary Study Objectives:

1. Assess the safety, tolerability, and acceptability of doxycycline PEP
2. Investigate the impact of doxycycline PEP on detection of culture and molecular
markers of tetracycline (TCN) resistance in C. trachomatis (CT), and T. pallidum
(syphilis- molecular markers only)
3. Evaluate the development of phenotypic tetracycline resistance among oropharyngeal
carriers of commensal Neisseria species
4. Measure the proportion of sex acts which are covered by doxycycline PEP
5. Assess the effect of doxycycline PEP on the gut resistome through measuring the
abundance of tetracycline resistance genes in stool of a subset of 50 MSM living with
HIV and 50 MSM/TGW on PrEP assigned to receive doxycycline PEP as well as rectal
swabs from all participants, stored for future evaluation

Exploratory Objectives:
1. Evaluate the diversity of the gut microbiome and quantity and breadth of drug
resistance genes in participants on doxycycline PEP and not on PEP
2. Assess the association between meningococcal vaccination and incident GC infection
3. Evaluate the incidence of nongonococcal, nonchlamydial urethritis and the proportion
of urethritis in which Mycoplasma genitalium (MG) is detected
4. Assess the rate of detection of Mycoplasma genitalium (MG) in urine in each arm and
the presence of tetracycline and other antimicrobial resistance in MG in each arm.
 39

5. Evaluate association of doxycycline use with weight change over time

6. Evaluate whether doxycycline PEP is associated with PrEP persistence and HIV
virologic suppression by comparison of arms and within the doxycycline PEP arm by
adherence to doxycycline PEP.
7. Evaluate the incidence of lymphogranuloma venereum (LGV) in those diagnosed
with rectal chlamydia in each arm

Approach: Eligible participants randomized to receive PEP will receive open-label doxycycline 200 mg
to be taken ideally within 24 hours but no later than 72 hours after each condomless sexual
contact (receptive anal, insertive anal, vaginal, or oral, or use of shared sex toys). No more
than 200 mg will be taken in each 24 hour period. Sexual activity and doxycycline PEP
usage will be recorded using a smartphone application and assessment at study visits. At
enrollment and quarterly for 12 months of follow-up, GC and CT testing will be conducted
on samples from the oropharynx, rectum and urine and a blood specimen for syphilis
serology will be collected. Resistance testing will be conducted on specimens that test
positive for GC using phenotypic methods, and molecular techniques for CT and syphilis
(for syphilis, DNA obtained from mucosal or lesional swabs). Nares and pharyngeal swabs
will be obtained to evaluate tetracycline resistance among nasopharyngeal carriers of S.
aureus and commensal Neisseria species (oropharyngeal swab only). Stool samples from
50 MSM/TGW living with HIV and 50 MSM/TGW on PrEP in the doxycycline PEP arm at 3
time points will undergo metagenomic sequencing for tetracycline resistance genes. Rectal
swabs from all participants who consent will be archived for future evaluation of the enteric
microbiome and resistome, as well as for presence of MG.

Study Schema
 40

1 BACKGROUND AND SIGNIFICANCE

1.1 Background
The incidence of bacterial sexually transmitted infection (STI) continues to increase in the US, with more than 2
million diagnoses of syphilis, gonorrhea (GC) and chlamydia (CT) in 2016.2 This STI epidemic is occurring
globally and is concentrated among men who have sex with men (MSM) and transgender women (TGW),
including those living with and without HIV. In the US, MSM accounted for 81% of syphilis infections in 2016 and
38% of gonorrhea cases; the latter represents a nearly 10-fold increase since 1989. 2 CT is also increasing
among MSM, with rectal chlamydia rates of 17% and the highest rates of CT diagnosed in STI clinics in MSM ≥
25 years old.2 Both MSM living with HIV and MSM without HIV infection on PrEP have very high bacterial STI
incidence, reaching or exceeding 50% new STI diagnoses per year.3, 4

Increasing STI rates have been associated with a rise in serious morbidity, including blindness secondary to
syphilitic ocular complications.5 Congenital syphilis rates have increased 28% in the past few years;6 it is likely
that some of this increase comes from ‘bridging’ from MSM to heterosexual populations.7 Distressingly, drug-
resistant GC continues to rise globally8-10 and in the US,11 leading to limited oral treatment options; recent cases
of highly-resistant GC in the UK and Australia required treatment with ertapenem.12, 13 Untreated STIs increase
the risk of HIV transmission from individuals with HIV infection who are not virologically suppressed,14, 15 as
occurs in nearly half of those living with HIV nationwide.16 With the recent CDC-endorsed “U=U” campaign (i.e.,
undetectable=untransmittable),17 condom use may further decrease among persons with HIV infection, and may
impact ‘serosorting’ in selection of sexual partners and decisions to use condoms. Accordingly, there is an urgent
need for innovative, effective, and acceptable interventions to halt the rise in syphilis, GC, and CT among MSM
and TGW living with and at-risk for HIV infection.

One potential STI control strategy for these high-risk populations is post-exposure prophylaxis (PEP)
with doxycycline. Antibiotic prophylaxis against STIs is not a new concept; minocycline was used as PEP for
GC in the US Navy in the 1940s and showed transient benefits with no clinical harms, but the rapid emergence
of tetracycline resistance prevented uptake of this approach as a public health strategy 18 A systematic review of
presumptive periodic treatment of sex workers showed overall 50% reductions in GC and CT without risk
compensation or increased AMR, and this approach is recommended for populations with high baseline GC and
CT prevalence 19. The rationale for STI prophylaxis among MSM and TGW is similar, given the high prevalence
and incidence of bacterial STIs among MSM living with HIV and MSM on PrEP. An effective STI prophylaxis
strategy could augment frequent STI screening, partner notification and other efforts to reduce STI acquisition
and transmission among MSM.
 41

Doxycycline has good tolerability, high bioavailability with oral Figure 1. IPERGAY Study: Time to first STI (panel a), GC
dosing, a 20 hour half-life, and excellent tissue penetration. infection (panel b), CT infection (panel c), and syphilis
infection (panel d) 1
Doxycycline is a first line treatment for CT with very high efficacy
(97-100%). Doxycycline is used to treat syphilis for persons with
penicillin allergy. Notably, CT and syphilis have not developed TCN
resistance in spite of decades of doxycycline use to treat these
infections. Doxycycline is not recommended for GC treatment due
to baseline resistance and increased TCN resistance will not affect
GC management.

The IPERGAY study demonstrated efficacy of single dose

doxycycline PEP after condomless sex among 232 HIV-negative
MSM taking event-driven PrEP in France.20 Doxycycline PEP
resulted in a 47% relative reduction in new bacterial STIs (GC, CT,
or syphilis) over 8.7 months of follow-up (45.9 vs. 79.6 per 100
person-years in those assigned to doxycycline PEP vs. no PEP
control, respectively; Figure 1A. Doxycycline PEP reduced the
incidence of CT and syphilis, but not GC (Figure 1B-D), which was
not surprising given doxycycline resistance in >50% of GC strains
in France. MSM in IPERGAY had a median age of 38 years, were
highly educated, majority Caucasian, and highly adherent to event-
driven HIV PrEP. MSM in the doxycycline arm in IPERGAY took a
median of 680 mg of doxycycline per month. Adverse events were
rare and there was no difference in self-reported sexual behavior
between MSM in the two study arms.

Limitations of generalizability from the IPERGAY study include that

the sample was small (n=232), and in contrast to MSM in the US
heavily impacted by STIs, MSM were older (median age 38), highly educated, and majority Caucasian. In addition,
with respect to adherence to event-driven doxycycline PEP, MSM in the IPERGAY study were highly adherent to
event-driven HIV PrEP and were already ‘cueing’ their TDF-FTC (Truvada) PrEP dosing to their sexual activity.
Importantly, uptake and adherence to doxycycline PEP among daily PrEP users are unknown. Additionally,
IPERGAY did not conduct detailed measurements of the pattern of doxycycline use with sexual practices nor
evaluate for antibiotic resistance associated with intermittent doxycycline use. Doxycycline PEP may be more
efficacious for reducing GC incidence in the US than in Europe, due to lower GC TCN resistance in the US (25%
vs 55% respectively).11, 20 PEP may also be more effective to prevent GC at non-pharyngeal sites, as evidenced
by the lack of an observed effect on pharyngeal GC incidence, whereas there was a trend toward reduction in
rectal and urethral GC infections with doxycycline PEP in IPERGAY.21

A small study piloted daily doxycycline among 30 MSM living with HIV with a history of syphilis, and showed a
73% reduction in composite STIs, without power to assess individual STIs.22 The greater risk of side effects and
antimicrobial resistance (AMR) with daily dosing of doxycycline have dampened enthusiasm for daily dosing of
doxycycline as PrEP against STIs. Event-driven doxycycline PEP has not been studied in MSM living with HIV.

Significant scientific knowledge gaps remain about on-demand STI doxycycline PEP in PrEP users and
MSM living with HIV. MSM in IPERGAY were already ‘cueing’ their Truvada PrEP dosing to sexual activity;
PEP adherence in daily PrEP users is unknown. IPERGAY did not conduct detailed measurements of the pattern
of doxycycline use with sexual practices nor report antibiotic resistance associated with intermittent doxycycline
use. Doxycycline PEP may be more efficacious for reducing GC incidence in the US than in Europe, due to lower
TCN resistance in GC the US (30% vs 55% respectively).11, 20 PEP may also be more effective to prevent GC at
non-pharyngeal sites; there was a trend toward reduction in rectal and urethral infections with doxycycline PEP
in IPERGAY.21 Notably, based on the IPERGAY demonstration of efficacy with doxycycline PEP, providers have
 42

begun to prescribe doxycycline PEP to reduce incident STIs in MSM without a confirmatory study or normative
guidance. Moreover, the balance of benefits and risks of doxycycline PEP need to be carefully studied in MSM
living with HIV as well as in PrEP users in a study powered to assess effectiveness for each population. The
relative benefits and risks of doxycycline PEP may differ in MSM living with HIV and PrEP users due to
differences in adherence, sexual practices, potential risk compensation, sexual networks, and/or pre-existing
antibiotic resistance due to higher prior antibiotic exposure among MSM living with HIV.

In summary, a sufficiently-powered, high-quality study is needed to evaluate the effectiveness of

doxycycline PEP in populations both with and without HIV infection, and to evaluate its impact on drug
resistance in target bacterial STIs and resident bacterial microbiota. There is a public health need and
sufficient clinical equipoise to conduct an effectiveness study of doxycycline PEP as an STI reduction strategy
for a diverse population of MSM in the US to address the following key scientific questions: 1) Effectiveness of
doxycycline PEP for bacterial STI prevention in MSM and TGW on PrEP, 2) Effectiveness of doxycycline PEP
in MSM and TGW living with HIV; 3) Differential tolerability and impact on antimicrobial resistance between
MSM/TGW living with HIV and MSM/TGW on PrEP; 4) TCN resistance in GC, CT and syphilis, 5) TCN resistance
in commensal Neisseria (which can transfer TCN resistance) and potentially pathogenic Staph Aureus in carriers,
and 6) effect on the gut resistome in terms of changes in TCN resistance genes.

1.2 Rationale
The overall goal of this study is to understand the effectiveness of doxycycline PEP on reducing STIs in MSM
and TGW populations at high risk for STI acquisition and to evaluate its impact on antibiotic resistance.

1.2.1 Doxycycline Safety

Several features of doxycycline make it an optimal choice for STI PEP. Doxycycline has been used safely and
for extended durations as prophylaxis against infections such as malaria23 and for non-infectious conditions
including rosacea24 and acne vulgaris.25 Doxycycline is generally safe and well-tolerated when used chronically.
Side effects include mild gastrointestinal symptoms, photosensitivity, and rarely pill esophagitis and pseudotumor
cerebri.26 Reassuringly, doxycycline has a much lower risk than other antibiotics for the antibiotic-associated
diarrhea caused by C. difficile infection,27, 28 and may even exert a protective effect against C. difficile.29, 30
Doxycycline is a scalable PEP intervention, as it is generic, inexpensive, and widely available. Doxycycline does
not require dose adjustment in hepatic or renal failure, including dialysis. In the IPERGAY doxycycline PEP study,
there was no difference in grade 3 or 4 adverse events between the study arms. As anticipated, there were more
gastrointestinal side effects in those on PEP (25%) vs. the non-PEP control arm (14%). Discontinuation of
doxycycline was uncommon with 8 study participants (7%) stopping doxycycyline due to side effects attributed
to doxycycline, the majority of which were gastrointestinal.1 Given the safety profile of doxycycline and the
enrollment of participants receiving ongoing clinical care for HIV or HIV PrEP, study-specific monitoring will be
limited to complete blood count and liver function tests at months 3 and 9 to assess for hematologic and hepatic
toxicity, which occur rarely with chronic doxycycline use (<1%). Assessment for side effects (eg., pill esophagitis,
photosensitivity, new onset headaches), with clinical and laboratory testing if indicated if potential serious
adverse events are suspected. Participants will receive standard of care monitoring through their HIV or PrEP.
Typical standard of care monitoring for PLHIV includes semi-annual viral load monitoring, renal and liver function
tests, and annual CBC. Typical standard of care monitoring for PrEP patients includes quarterly HIV tests and
semi-annual or annual renal monitoring.

1.2.2 Rationale for single dose doxycycline as STI PEP.

A concern about a single post-sex dose of doxycycline is the long in vivo doubling time of syphilis (approximately
30-33 hours)31, 32, which is the rationale for treatment of early syphilis with long-acting IM penicillin or oral
doxycycline for 14 days. However, the incubation time for syphilis is 10-90 days,33and doxycycline PEP
administered within 72 hours of exposure may deliver sufficient chemoprophylaxis to abort primary infection.
Notably, in IPERGAY, a 73% reduction in syphilis incidence was observed with single dose doxycycline.1 In
 43

addition, single dose doxycycline PEP has been shown to be effective for Lyme disease34 and leptospirosis 35, 36
– spirochetal diseases that also have slow doubling times.35, 37 An alternative approach is doxycycline PrEP (i.e.,
daily dosing).22 However, daily doxycycline PrEP may have a higher frequency of intolerability due to side effects,
selection of antimicrobial resistance through higher exposure, and greater costs than doxycycline PEP.
Importantly, MSM in IPERGAY demonstrated their ability to effectively adhere to peri-coital Truvada PrEP and
post-coital doxycycline PEP.

1.2.3 Potential for drug resistance

An important consideration with intermittent doxycycline use for STI prophylaxis is selection of drug resistance,
in both target bacterial STI pathogens as well as in microbiota which are potential pathogens or possible
reservoirs of antimicrobial resistance, such as S. aureus and commensal Neisseria species. GC has sequentially
developed resistance to antibiotics in multiple classes, including those previously or currently recommended for
treatment. Rates of TCN-resistance in GC in the US are 25%-38% in studies of MSM,11 compared with >50% in
Europe.20 Doxycycline is not currently recommended as treatment for GC. IPERGAY has not reported on TCN
resistance in GC and did not address AMR in other bacterial such as S. aureus and commensal Neisseria
species. Doxycycline is commonly used to treat CT and syphilis, and there have been no documented cases of
TCN-resistant CT or syphilis. However, TCN resistance from single point mutations has been reported in related
organisms such as Chlamydia suis38, 39 and Brachyspira spp.40 in antibiotic-exposed pigs, suggesting that
development of TCN resistance could evolve (or be acquired from a resistant organism such as E. coli 41).
Azithromycin-resistant Treponema pallidum emerged from a single mutation42 and rapidly became widespread
in the US west coast and globally,43 serving as a cautionary tale.

Repeated antibiotic exposure can foster drug resistance in the normal commensal flora of the body and impact
the composition of gut microbiome. The effect of doxycycline use on S. aureus is an important consideration,
given that ∼30% of the population are S aureus carriers.44 AMR is concerning for community-based MRSA
infections, for which doxycycline is sometimes used as an alternative to sulfa drugs.45 Similarly, doxycycline
exposure could also drive development of TCN resistance in commensal Neisseria species, serving as a
reservoir that potentially can transfer resistance to GC. Antibiotics affect the larger microbiome, with decreased
diversity of gut flora and shifts in the predominant species, which may fuel a “dysbiotic” environment and may
contribute to inflammation and complications such as C. difficile.46-48 In sum, a comprehensive assessment of
doxycycline PEP is needed, including public health benefits, safety, tolerability, adherence, and AMR among
bacterial STIs and organisms that may can be pathogenic (e.g. S. aureus among carriers) or transfer resistance
to GC (e.g., from commensal Neisseria).

1.2.4 Rationale for evaluation of meningococcal vaccination status

A recent report from New Zealand suggests that vaccination with an outer membrane-based Group B Neisseria
meningitidis vaccine is associated with a reduced risk of gonorrhea in adolescents and adults aged 15-30 years
of age; the estimated effectiveness of the OMV meningococcal vaccine against gonococcal infection to be 33%
[ NZ OMV is part of the composition of the 4CMenB vaccine, commercially distributed as BEXSERO49 (GSK). It
is not known if other meningococcal vaccines are associated with a similar decrease in GC infections.
Meningococcal vaccination (Serogroup A/C/W/Y) is currently recommended by the ACIP for all PLWH and
serogroup B vaccination is a consideration for adolescents and young adults50. Meningococcal vaccine status
will be recorded, with the type of vaccine if known, to evaluate for a potential association of vaccination with
decreased GC rates.
 44

1.2.5 Rationale for evaluation of nongonoccal urethritis (NGU) and Mycoplasma genitalium (MG)
Mycoplasma genitalium is a frequent cause of urethritis in men .51 MG can be detected with nucleic acid testing;
however, many public health programs do not routinely test for MG and instead treat non-gonoccocal urethritis
(NGU) empirically.52 While azithromycin is superior to doxycycline for treatment of MG, 53, 54 doxycycline does
have activity against MG and use of doxycycline PEP may potentially impact the prevalence of MG, the incidence
of MG-associated urethritis and the syndrome of NGU. Therefore, urine will be collected every three months to
look for the presence of asymptomatic MG as well as at the time of urethral symptoms, to evaluate for MG as
the etiology of urethritis.53, 54 Specimens with MG detected will be stored for future analysis of resistance to
tetracycline and other antimicrobials.

1.2.6 Rationale for evaluation of longitudinal weight change

Tetracyclines have been associated with weight gain in mice,55 as well as in individuals taking tetracycline-
based combination therapy for Q fever56 and for Helicobacter pylori.57, 58 Therefore, participants in both arms
will have weight evaluated at regular intervals to evaluate for possible weight gain

1.2.7 Impact of COVID-19 on study conduct

Study enrollment and the majority of in-person study evaluations were halted in March 2020 due to the COVID-
19 pandemic and the hold on clinical research required by the participating institutions. As a result, patients
enrolled 3/2020 or earlier were unable to undergo regular ascertainment for the STI endpoint during the following
months. Given the need for full endpoint ascertainment and acknowledging potential changes in sexual behavior
during the COVID pandemic, participants enrolled 3/2020 and earlier will be offered a full 12 months of follow up
at the time of resumption of follow up after COVID restrictions are lifted. Those who decline a 12 month follow
up will be discontinued. The team will monitor discontinuation rates in this pre-COVID cohort. If there is a
differential discontinuation in the active vs. control arm, this will be discussed with DSMB and the team statistician
with consideration of additional enrollments to ensure adequate power for the primary endpoint

As COVID may continue to impact research activity in the future, the study will allow for electronic consenting
as an option, and shipment of doxycycline to participant homes, if necessary and if acceptable to study
participants. In-person visits will be preferred when feasible to allow for STI testing, research labs, and pill
counts. Home collection of STI testing may be considered, as long as there is sufficient data to support the
testing platforms for use in home collection, particularly in terms of syphilis testing.

1.2.8 Early closure by the DSMB and revision to study conduct

Enrollment into the DoxyPEP study was stopped early on 5/13/2022, as a prespecified interim data analysis
demonstrated that the study had crossed pre-specified effectiveness thresholds. At the time of interimr review
of 554 participants enrolled, a single dose of 200 mg doxycycline taken after condomless sex significantly
reduced the acquisition of gonorrhea, chlamydia and syphilis in men who have sex with men (MSM) and
transgender women (TGW). The effectiveness of doxycycline PEP was observed and statistically significant
both in the cohort of participants living with HIV and the cohort of participants without HIV who were taking
PrEP. The strategy of doxyPEP was safe and well-tolerated, with no doxycycline-associated Grade 2 or higher
adverse events and no doxycycline-associated serious adverse events.

Enrolled participants will be notified of the study early closure and of the finding of doxycycline effectiveness
to reduce STIs. They will also be informed that the impact of doxycycline PEP on antimicrobial resistance in
STIs, S. aureus, commensal Neisseria and the gut microbiome is still under investigation. This protocol
modification amends the study to offer doxycycline to enrolled participants assigned to the standard of care
arm for the remainder of their 12 month participation. Participants who roll over from the control arm into the
active doxycycline PEP arm will undergo the same evaluations as specified for the active arm (e.g., safety
labs) per the schedule of events and will continue with quarterly study visits through month 12. At the time of
roll over onto active doxycycline PEP, a rectal swab to establish the microbiome baseline prior to starting
doxycycline will be collected. Participants who decline to roll over into the active doxycycline arm will have the
option of remaining on study through month 12 following the standard of care arm schedule of events. All
 45

participants will review and sign a revised informed consent at the time of their next study visit to consent to
continuation in the study and to receive doxycycline, for those in control arm who chose to roll over to active
doxyPEP.

The goal of continued follow-up through Month 12 of enrolled participants receiving doxycycline PEP is to
obtain additional data on the tolerability and acceptability of doxycycline PEP and the impact of intermittent
doxycycline PEP on antimicrobial resistance and the gut microbiome. This information is critical for
understanding the risk/benefit profile of doxycycline PEP and to inform guidelines about the doxyPEP strategy
which will be considered both by individuals, providers, and public health authorities.

2 STUDY DESIGN

Design: The overarching goal is to assess the effectiveness of doxycycline PEP on incidence of STIs and
tetracycline resistance among STIs and commensal bacteria to inform public health policy. Participants will be
randomized 2:1 (see Schema), with two-thirds receiving doxycycline PEP and one-third receiving the standard
of care control, to maximize data on safety, tolerability, adherence coverage of sexual acts, and resistance data
in participants randomized to doxycycline PEP, without negatively impacting power to measure effectiveness.
Participants will be counseled about the preliminary effectiveness data from IPERGAY, and the potential for AMR
in STIs or other bacteria. Possibility of unreported doxycycline use in the control arm (contamination) will be
monitored through retrospective batch testing of doxycycline metabolites in hair, to detect doxycycline use in the
prior 3 months. 59

The trial of doxycycline PEP will be powered to separately assess impact for MSM living with HIV and MSM on
PrEP because of potential differences in safety, tolerability, adherence, sexual networks, sexual practices,
background AMR, and ultimately, PEP effectiveness. Eligible participants randomized 2:1 to receive PEP will
receive open-label doxycycline 200 mg to be taken ideally within 24 hours but no later than 72 hours after
condomless sexual contact (oral or anal). 200 mg of doxycycline will be taken at most once per 24 hour period
regardless of the number of sexual acts occurring during this time period. the rationale for 2:1 randomization is
to maximize data on doxycycline PEP safety, tolerability, acceptability, and resistance, while providing sufficient
power for the primary outcome of the effectiveness of doxycycline PEP. Sexual activity will be recorded for both
arms of the study (doxycycline PEP and control condition) by the participant using a smartphone application that
will be adapted for study use; this will enable comparable assessment of risk in the two arms. PEP pill-taking will
also be measured by the app to enable assessment of coverage of sex acts by PEP. Sexual activity and
adherence will also be assessed in person at quarterly visits. STI testing will be conducted quarterly from three
anatomic sites (pharyngeal, rectal, and urinary) and blood obtained for syphilis testing, following CDC guidelines
for serologic diagnosis of syphilis. Participants with a positive STI test will return for STI treatment and for swabs
of the affected site for resistance testing; culture based for GC and molecular methods for CT and syphilis. Those
with signs and symptoms suggestive of syphilis infection and those with a reactive syphilis serologic test that
indicates a new syphilis infection will have swabs of any current active lesion (if present) as well as mucosal
swabs from the oropharynx. Swabs from the anterior nares and oropharynx will be obtained at baseline, 6 and
12 months to evaluate tetracycline resistance in S. aureus among carriers and oropharyngeal swabs will be
obtained at baseline and 12 months to evaluate tetracycline resistance in commensal Neisseria species.

Stool samples from 100 participants on the doxycycline PEP arm − 50 MSM and TGW living with HIV and 50
MSM and TGW on PrEP− will be collected at baseline, 6 and 12 months to evaluate effects of intermittent
doxycycline on the gut resistome, using FLASH targeted metagenomic sequencing to evaluate for tetracycline
and other resistance genes. Rectal swabs will be collected and archived in all participants at baseline, 6, and 12
months for future studies of the impact of doxycycline PEP on the enteric microbiome and resistome.

Study population: This study will enroll 780 participants living with HIV and HIV-negative persons taking PrEP.
An approximately equal number of PLWH and HIV uninfected persons taking PrEP will be enrolled. An
approximately equal number of participants in each of these cohorts (and in each study arm) will be enrolled in
 46

San Francisco and Seattle.

Current or planned initiation of PrEP use is an eligibility criterion for enrollment, because this population of MSM
and TGW has high rates of STIs and is typically seen quarterly for PrEP visits. However, participants may opt to
stop PrEP use at any time during the study without affecting their involvement in the study. Any participants
without HIV infection who subsequently seroconvert will be managed clinically by the study site according to
local practice (appropriate counseling, clinical evaluation, and immediate linkage to clinical and psychosocial
services). These participants will also be retained in the study unless they choose to discontinue study
participation.

3 OBJECTIVES

3.1 Primary Objectives

3.1.1 Evaluate the effectiveness of doxycycline post-exposure prophylaxis (PEP) to reduce STI infections
in two populations: a) MSM/TGW living with HIV and b) MSM/TGW taking HIV PrEP.
3.1.2 Investigate the impact of doxycycline PEP on development of culture-based tetracycline (TCN)
resistance in N. gonorrheae and among nasopharyngeal carriers of S. aureus

3.2 Secondary Objectives

3.2.1 Assess the safety, tolerability, and acceptability of doxycycline PEP
3.2.2 Investigate the impact of doxycycline PEP on detection of culture and molecular markers of tetracycline
(TCN) resistance in C. trachomatis (CT), and T. pallidum (syphilis- molecular markers only).
3.2.3 Measure the proportion of sex acts which are covered by doxycycline PEP
3.2.4 Evaluate the development of phenotypic TCN-resistance among oropharyngeal carriers of commensal
Neisseria species
3.2.5 Assess the effect of doxycycline PEP on the gut resistome through measuring the abundance of
tetracycline resistance genes in stool of a subset of 50 MSM living with HIV and 50 MSM/TGW
on PrEP assigned to receive doxycycline PEP as well as rectal swabs from all participants,
stored for future evaluation

3.3 Exploratory objectives

3.3.1 Evaluate the diversity of the gut microbiome and quantity and breadth of drug resistance genes
in participants on doxycycline PEP and not on PEP
3.3.2 Assess the association between meningococcal vaccination and incident GC infection
3.3.3 Evaluate the incidence of urethritis without identified bacterial etiology and the proportion which is
associated with MG in each arm

3.3.4 Assess the rate of detection of Mycoplasma genitalium (MG) in urine in each arm and the
presence of tetracycline and other antimicrobial resistance in MG in each arm.
3.3.5 Evaluate association of doxycycline use with weight change over time
3.3.6 Evaluate whether doxycycline PEP is associated with PrEP persistence and HIV virologic
suppression by comparison of arms and within the doxycycline PEP arm by adherence to
doxycycline PEP.
3.3.7 Evaluate the incidence of lymphogranuloma venereum (LGV) in those diagnosed with rectal
chlamydia in each arm

4 SELECTION AND ENROLLMENT OF PARTICIPANTS

 47

4.1 Inclusion criteria

4.1.1 Willing and able to give written informed consent
4.1.2 Age > 18 years
4.1.3 Male sex at birth
4.1.4 Previously HIV-diagnosed
OR
HIV-seronegative at the time of last test within the past three months and a current prescription for
PrEP (both daily or event-driven PrEP permitted) or plan to start PrEP within 30 days after the
enrollment visit
4.1.5 Condomless anal or oral sexual contact with ≥ 1 male sex-at-birth partners in the past 12 months
4.1.6 Diagnosed with GC, CT or syphilis in the past 12 months.
Note: self report of STI is acceptable if documentation not available. If the participant reports an incident
STI in the past year at the same clinic where the participant will be enrolled, this diagnosis should be
confirmed by chart review prior to enrollment. If the diagnosis from this clinic cannot be confirmed, the
participant should not be enrolled. If the STI was reported at a clinical site that is not the study site, and
records cannot be obtained, self-report will suffice.

Note: Syphilis diagnosis within the last year refers to primary syphilis, secondary syphilis, and
documented early latent syphilis (< 1 year since last syphilis diagnosis or negative test). Known late
latent syphilis or latent syphilis of unknown duration would not qualify. Positive syphilis titers which
represent serofast status and not active disease do not qualify as a syphilis diagnosis. Clinician
judgement regarding qualifying syphilis diagnosis should be sought when the diagnosis of syphilis in the
past year is not clear or if there is a question about serofast status vs. active infection.

4.2 Exclusion criteria

4.2.1 Allergy to tetracycline class
4.2.2 Current medications which may impact doxycycline metabolism or that are contraindicated with
doxycycline, as per the prescribing information. These include systemic retinoids, barbiturates,
carbamazepine, and phenytoin.
4.2.3 Current use of warfarin, as intermittent doxycycline use can lead to an unpredictable impact on
INR
4.2.4 Anticipated use of doxycycline during the coming 12 months for non-STI prevention use (e.g.,
acne treatment).

4.3 Recruitment
Each site has established local recruitment and screening methods that operationalize protocol-specified
requirements for eligibility determination in a manner that is tailored to and most efficient for the local study
setting and target study population. Each site will use a variety of recruitment approaches, including direct
recruitment at clinics, referrals from other providers of PrEP and ART, and use of online and social networking
websites and apps. Recruitment materials will educate participants about STI prevalence and incidence in their
community and the pilot study that demonstrated efficacy of doxycycline for reducing incidence of STIs among
MSM in IPERGAY.

The study will enroll from 4 clinical sites, 2 in Seattle and 2 in San Francisco (Table 1), which have established
track records of high quality research in MSM and TGW populations integrated into clinical care settings;
annual retention rates in clinical trials conducted in these sites exceed 80%-90%. The sites have large MSM
populations that are ethnically and racially diverse.

Table 1. Study Sites

 48

High volume, comprehensive HIV care clinics with • “Ward 86” HIV Clinic, ZSFG, UCSF, San
on-site STI testing & treatment. Francsico, CA
• Madison (HIV) Clinic, Harborview Medical
Center, UW, Seattle, WA
Municipal STI clinics with integrated on-site HIV • San Francisco City Clinic, San Francisco CA
PrEP programs. • Public Health – Seattle & King County
Sexual Health Clinic, Seattle, WA

4.4 Co-enrollment guidelines.

Participants may be co-enrolled in other research studies, provided that these are a) observational studies or b)
evaluation of an FDA approved intervention which is not an antibiotic, is not intended to treat or prevent an STI,
and is compatible with coadministration of doxycycline. The study team should be consulted for co-enrollment
in studies that do not meet this guidance or if there are questions about eligibility for co-enrollment. For any co-
enrolled study, combined blood draws should not exceed current Red Cross phlebotomy guidance.

5 STUDY TREATMENT

5.1 Study product

Generic delayed release doxcycyline hyclate 200 mg capsules (Mayne Pharmaceuticals) will be provided by
the study to participants randomized to open-label PEP, and to participants who choose to roll over to active
doxycycline PEP following the 5/13/2022 closure of enrollment. Doxycycline DR is bioequivalent to
doxycycline hyclate IR. 60, 61 Doxycycline hyclate is widely available and FDA approved for the treatment of
multiple infectious conditions. The full prescribing information can be accessed at:
https://dailymed.nlm.nih.gov/dailymed/drugInfo.cfm?setid=cd2ab9b8-9619-4199-8a5d-83377b3274d1

5.2 Safety of doxycycline

Doxycycline is an antibiotic that can be used for prolonged periods of several months in the treatment of acne or
in the prevention of malaria. The tolerance of repeated-dose of doxycycline in this 12 months study should
therefore be acceptable.

The most common side effects of doxycycline are62, 63

• Gastrointestinal: nausea, diarrhea, epigastric pain, anorexia, glossitis, enterocolitis, anal and genital
candidiasis
• Esophageal disorders: dysphagia, pill esophagitis, and rarely esophageal ulceration. Risk for
esophageal irritation can be reduced by taking doxycycline tablets at least one hour before bedtime (to
avoid lying down during doxycycline intake) at a meal with a large glass of water (100 ml).
• Skin reactions including maculopapular, erythematous and photosensitivity skin reactions. Due to
concern for photosensitivity, individuals taking doxycycline are advised to avoid excessive exposure to
sunlight and UV radiation (such as tanning beds).

Other possible but rare adverse effects occurring in < 1% in post-marketing reports include:
• Hypersensitivity reactions: urticarial, anaphylaxis, serum sickness, drug reaction with eosinophilia and
systemic symptoms (DRESS)
• Skin toxicity: fixed drug eruption. Stevens-Johnson syndrome, toxic epidermal necrolysis, erythema
multiforme, fixed drug eruptions exacerbation of preexisting lupus erythematosus
• Pericarditis
• Hematological disorders: hemolytic anemia, thrombocytopenia, neutropenia, eosinophilia
• Intracranial hypertension (also known as pseudotumor cerebri)
 49

5.3 Doxycycline dispensing and administration.

Doxycycline will be dispensed at each 3-month visit or in shorter intervals per patient preference, with additional
dispensation as needed to replace lost medication or provide additional medication when needed (see MOPS
for addition dispensing guidance). Capsules should be stored at room temperature (59-86o F) to the extent
feasible. Participants will be instructed to take 200 mg of doxycycline within 24 hours after condomless sexual
contact, and no later than 72 hours after sex. Condomless sexual contact is defined as receptive anal, insertive
anal, vaginal, or oral sex (which includes oral/anal and oral/genital contact) or use of shared sex toys without a
condom used for the entire time. For participants reporting multiple sexual encounters, 200 mg of doxycycline
will be recommended to be taken every 24 hours until after the last sexual encounter. Participants will be
instructed not to exceed 200 mg of doxycycline in a 24 hour period. Study staff will instruct participants on
procedures for replacement of lost medication.

Doxycycline may be taken on an empty stomach or with food, and it is advised to take each dose with a large glass
of water at least 60 minutes before bed to reduce risk of esophageal irritation. All participants randomized to PEP
will be advised to avoid excessive sun exposure and artificial UV light, such as tanning beds, and to consider
use of sunscreen if they will be exposed to sun.

5.4 Concomitant medications

Participants randomized to doxycycline PEP should report current and new medications to the study team to
ensure no concern for drug interactions with doxycycline. The prescribing information for doxycycline
monohydrate should be reviewed to ensure no potential for drug interactions.

5.4.1 Prohibited medications

• Barbiturates
• Carbamazapine
• Phenytoin
• Methoxyflurane
• Systemic retinoids, such as acitretin and isotretinoin

5.4.2 Precautionary Medications

• Antacids containing aluminum, iron, calcium, or magnesium, or bismuth subsalicyclate products
(such as Pepto-Bismol) may impair doxycycline absorption and should be separated 2 hours
before or after doxycycline
• Oral iron supplements may impact doxycycline absorption. Iron should be separated 2 hours
before or after doxycycline

5.5 HIV pre-exposure prophylaxis (PrEP) and HIV antiretroviral therapy (ART)
PrEP and ART will not be provided by the study but will be accessible through local providers following the
standard of care. Each of the participating sites has a clinical infrastructure in place to assist individuals with
obtaining ART and PrEP, including for those without insurance or whose insurance status changes.
 50

6 CLINICAL AND LABORATORY MONITORING

6.1 Schedule of Events

Doxycycline
12 or
initiation visit for
Month (+2/-10 week window) 0 3 6 9 Termination
those in control
visit
arm
Informed consent √ √
Smartphone app instructions √
Review of current medications √ √* √* √* √* √
Antibiotic & STI history √ √ √ √ √
Meningococcal vaccination history √ √
Sexual behavior questionnaire √ √ √ √ √
Dispense pills/adherence counseling* √* √* √* √* √
Doxycycline pill count and adherence
√* √* √* √*
recall*
Doxycycline PEP acceptability survey* √* √*
General symptom assessment √ √ √ √ √
Costing questionnaire √ √ √ √
Condoms/lubricant √ √ √ √ √
Risk reduction counseling √ √ √ √ √
GC/CT testing (throat & rectal swab, urine
√ √ √ √ √
collection)
Syphilis testing √ √ √ √ √
Rectal swabs stored for future microbiome
√ √ √ √
testing, all participants
Rectal swab stored for future MG testing √ √ √ √ √
Urine stored for future MG testing (also at
√ √ √ √ √
time of symptomatic urethritis)
Swabs/urine at time of evaluation for STI
symptoms or confirmed STIs for at time of evaluation for STI symptoms or confirmed STI
resistance testing
Anterior nares and oropharyngeal swabs
√ √ √
for Staphylococcus aureus culture
Oropharyngeal swabs for cultures of
√ √
Neisseria species
Stool sample from first 50 PLWH & first 50
√* √* √*
HIV- on PrEP*
Collection of hair for doxycycline testing √ √
 51

HIV testing data from local care

√ √
(HIV RNA, CD4)
CBC and liver function tests* √* √* √**

Stored serum for future testing (Optional) √ √

Reimbursement √ √ √ √ √
Weight √ √ √ √ √
Qualitative interviews*
3-12 months
(subset of participants, optional)
*Intervention arm only **Standard of care arm only
 52

6.2 Study visits

Specific study procedures are detailed in Table 6.1. Visits will take place at enrollment and quarterly thereafter,
up to 12 months. Interim visits may be scheduled for diagnosis and treatment of STI and collection of additional
specimens or for evaluation of possible doxycycline associated adverse events. Participants may also request
interim STI testing.

6.2.1 Enrollment Visit After confirming eligibility through pre-screening and prior to study procedures, we will
obtain written informed consent and collect locating information; the latter will be updated at each study
visit. At enrollment, demographic, behavioral, and clinical information will be collected and a release of
information obtained to permit access to medical records. STI testing for chlamydia and gonorrhea
through nucleic acid amplification testing (NAAT) of clinician or self-collected pharyngeal and rectal
swabs and first void urine will be conducted. Serologic testing for syphilis will be conducted. If required
STI testing has been conducted within past 30 days, these results may be used for the enrollment
evaluation. Participants diagnosed with an STI at baseline will be treated; presence of a baseline STI
will not preclude enrollment but STI’s present at time of enrollment will not count towards study
endpoints. All STIs will be reported to the local health department as required per local standard
procedures.

Randomization Within 14 days of enrollment, participants will be randomized in a 2:1 ratio to open-
label doxycycline PEP or the standard of care control condition (regular STI counseling, screening and
treatment as indicated). The randomization code and resulting allocation list will be generated and
maintained by the study statistician. The list will be blocked and stratified by site. While neither
participants nor study staff will be blind to each participant’s randomization group once assigned, the
randomization scheme will utilize varying block sizes in order to protect the blind prior to randomization
of each participant.

6.2.2 Quarterly visits after enrollment. Quarterly visits will be conducted every 90 days, +2 weeks/ -10
weeks. Participants unable to attend a quarterly visit will be asked to return for an unscheduled visit as
soon as feasible. The quarterly evaluations should be completed at the time of this visit. Subsequent
visits should occur on the assigned quarterly scheduled to the extent possible.

6.2.3 Unscheduled interim visits: Participants will be instructed to contact the study site and return for an
unscheduled visit for any of the following a) new diagnosis of STI, b) concern for possible adverse event
associated with doxycycline, c) need for additional doxycycline (in participants who were randomized
to PEP arm), d) difficulty using the smartphone app, or any other study-related concerns.
Reimbursement will not be provided for unscheduled visits. All interim contacts and visits will be
documented in participants' study records and on applicable CRFs.

6.2.4 Termination visit: In the event that a participant prematurely discontinues study participation, the
evaluations for the termination visit should be completed if possible.

6.3 Instructions for evaluations

6.3.1 Smart phone application: A smartphone application (app), Blackbook, (developed in conjunction
with UCSF researchers) currently in use to track sexual behavior and PrEP use in young MSM has
been adapted for use in this study to measure daily sexual risk practices (both study arms) and
doxycycline PEP administration (intervention arm only). Study staff will assist with obtaining
smartphones through local available resources such as Lifeline Assist, which make cell phones
available to qualifying lower income individuals, when needed and feasible
(https://www.truconnect.com/lifeline/freephone/signup). Participants will be given unique codes to
permit download of the app from an online resource and guidance on how to use the application.
 53

6.3.2 Review of current medications: All participants will have current medications reviewed at screening
for possible drug interaction with doxycycline. At subsequent quarterly visits, participants randomized
to doxycycline PEP will have current medications be reviewed and any new medications will be
assessed for possible drug interactions.
6.3.3 STI symptom screen. Participants will be asked about current STI symptoms including dysuria, rash,
throat pain, rectal pain/discharge, and genital lesions
6.3.4 Symptom assessment. At each visit, participants in both study arms will be asked about possible
doxycycline side effects, including esophagitis, rash, gastrointestinal symptoms, photosensitivity,
headache, and changes in vision (including blurred, double vision, vision loss). Control arm
participants will be asked about these symptoms to establish a comparator for non-specific symptoms
which can occur in the absence of doxycycline.
6.3.5 Antibiotic and STI history: History will be recorded of any non-study provided antibiotics taken in the
last 3 months and any STI diagnosis in preceding 3 months, including the diagnosis of urethritis
requiring treatment but without detection of gonorrhea or chlamydia. At study entry, STI history for
preceding 12 months will be recorded as well as obtained through chart review when possible.
6.3.6 Meningococcal vaccination history: At study entry and at month 12, receipt of any prior
meningococcal vaccination and the type (if known) will be recorded, using a checklist that will include
- Serogroup A only (includes MenAfriVa, PsA-TT)
- Serogroup B only (includes Trumenba, Bexsero)
- Serogroup C only (includes Meningitec, NeisVac C)
- Quadrivalent A/C/Y/W (includes Menomune, Menveo,Nimirix, Menactra)
- Or other formulation
Type of vaccination may be confirmed by chart review if records are available.
6.3.7 Sexual behavior questionnaire: Participants will be asked to complete a sexual behavior
questionnaire recording number and type of sexual contacts in the past 3 months at enrollment and
between visits
6.3.8 Pill count and doxycycline adherence assessment. Participants assigned to doxycycline PEP or
who roll over to receive doxycycline in the control arm will be asked to bring their doxycycline bottles
in at each visit for a pill count. They will be asked what proportion of sex acts were covered by PEP
in the past month and past 3 months, an estimate of overall adherence, and how many total doses of
doxycycline were taken in the past 3 months.

Note: any period of 7 or more days in which the participant had condomless sexual activity but did
not take doxycycline will be recorded as a doxycycline interruption. This includes interruptions due to
lack of doxycycline, forgetting to take doxycycline, taking medication not compatible with doxycycline,
or participant decision to temporarily or permanently discontinue study-provided doxycycline.
6.3.9 Doxycycline PEP acceptability survey. At months 6 and 12, participants assigned to or now taking
doxycycline PEP will be given a brief self-administered questionnaire evaluating acceptability of PEP
6.3.10 Risk reduction counseling and offer to provide of condoms/lubricant. At each visit, staff will
counsel all participants about STI risk reduction and HIV prevention (if not living with HIV), and the
importance of adherence to ART and PrEP, according to local standard of care.
6.3.11 STI testing.
GC/CT: First void urine and pharyngeal and rectal swabs will be collected for NAAT testing. Swabs
may be self collected or collected by study staff. GC & CT testing should be conducted at least 14
days after treatment to avoid false positive nucleic acid results.

Note: If GC or CT have been conducted within 30 days prior to study visit and these results are
available, these results may be used for the quarterly STI assessment. All available STI results should
be recorded.

RPR: Blood will be collected for non-treponemal assay (RPR or VDRL) with titer if positive.
Treponemal assay results should be recorded if RPR or VDRL are positive. Syphilis testing will be
 54

repeated quarterly after last treatment for syphilis to determine if titers fall or if not, for clinical
evaluation of treatment failure or reinfection. Titers must be interpreted in the context of prior test
results and current symptoms and exposure history. The need for treatment will be based on
the clinical assessment of the site investigator based on the serological data and clinical findings. All
syphilis endpoints will be determined by the Endpoint Adjudication committee (see 10.3)

Note: It is preferred to not repeat syphilis testing at a quarterly visit if testing has been done less than
12 weeks before the visit and these tests are available. All available syphilis test results should be
recorded.

6.3.12 Rectal swab for future M. genitalium and other testing.

Rectal swab will be collected and stored for future batched M.gen testing, including M.gen antibiotic
resistance testing, and potential testing for other infectious organisms. Swabs may be self-collected
or collected by study staff.

6.3.13 Urine for M.genitalium testing

At the time of urine collection for GC/CT testing, a sample of this urine collection will be stored for
future batched testing for MG. Remnant urine from GC/CT testing may be used or the initial first catch
specimen may be divided into two samples, as long as the minimum volume for GC/CT testing is met.
See the MOPS for more information. Urine should also be collected for M.gen at the time of
symptomatic urethritis (see Table 6.4.1)
6.3.14 Nasal and oropharyngeal swab for Staphylococcus aureus: nares and oropharynx will be swabbed
and cultured for S. aureus, with antibiotic resistance testing if S. aureus growth present
6.3.15 Oropharyngeal swab for commensal Neisseria species. At baseline and month 12, all participants
will undergo oropharyngeal swab for Neisseria species and TCN-resistance testing if Neisseria
species are isolated.
6.3.16 Stool specimen: Stool samples from the first 50 participants living with HIV and 50 HIV- participants
on PrEP who consent to collection will be obtained at baseline, 6 and 12 months for metagenomic
resistome studies using 16sRNA amplification of TCN resistance genes. In the event that participants
cannot provide a stool sample at the scheduled visit, they will have up to 2 weeks from the enrollment
and 12-month visit to return the stool specimens for those visits, and up until two weeks after the 9-
month visit to return the stool specimen for the 6-month visit.
6.3.17 Rectal Swab for microbiome testing: Rectal swabs will be collected and frozen for future
microbiome analysis. These swabs may be self collected or collected by study staff.
6.3.18 Hair collection: Hair will be collected from all participants who consent to collection and who meet
eligibility for hair collection (occipital hair length > 0.5 cm and absence of bleaching in area to be
collected) See manual of procedures for details on hair collection
6.3.19 HIV testing and RNA data:
For PLWH: At entry and month 12, the most recent HIV RNA data available from clinical visits will be
recorded
For participants on PrEP: At entry, the most recent HIV antibody or viral test results from clinical care
will be recorded. If no test results available within the past 3 months, HIV testing should be obtained,
consistent with standard of care monitoring for PrEP. At month 12, the most recent HIV antibody or
viral load test results from clinical care will be recorded.
6.3.20 ART and PrEP status
For PLWH, current ART regimen and status (taking vs. not taking) will be recorded at each visit. At
entry and month 12 for PLWH, the most recent HIV RNA and CD4+ cell count available from clinical
visits will be recorded if available
For participants on PrEP At each visit for those on PrEP, PrEP status will be assessed (taking vs.
not taking, and how PrEP is taken- daily vs intermittent ). At entry and month 12, the most recent HIV
antibody or viral load test results from clinical care will be recorded.
6.3.21 Hematologic and liver function tests: Complete blood count (CBC) (white count with differential,
hemoglobin, platelets) and liver function testing (AST, ALT, total bilirubin and alkaline phosphatase)
 55

will be drawn at months 3 and 9 in those on doxycycline PEP. Testing done through standard of care
within 30 days of the study visit may also be used if available. For those in the standard of care arm,
CBC will be done at the month 12 visit. In the event that a CBC cannot be obtained at month 12, an
available CBC from any time during study participation may be used.
6.3.22 Serum for storage (Optional): At months 0 and 12, 10 mL of serum will be collected for storage for
future analysis (non-genetic) in participants who agreed to this. Serum may be collected up to 14
days after the visit if blood cannot be collected at the time of the visit.
6.3.23 Reimbursement: Participants will be reimbursed at the enrollment visit, at quarterly visits, and at the
time of return for treatment of STIs with accompanying collection of swabs. An additional
reimbursement will be provided for participants who consent to the optional stool sample collection at
the baseline, 6 month, and 12 month timepoints. No reimbursement will be provided at other
unscheduled interim visits such as visits to provide additional doxycycline.
6.3.24 Roll over from standard of care to active doxycycline: All participants initially assigned to the
standard care arm will be offered the option to receive doxycycline through the study for the remainder
of their 12 month follow up period. If a standard of care arm participant elects to initiate doxycycline,
they will complete a new informed consent at the doxycycline initiation visit, and will be provided with
instructions and counseling on how to use doxycycline PEP. If a control arm participant comes in to
initiate doxycycline at a time that falls within the visit window for the next scheduled study visit, all
scheduled visit evaluations will be conducted, in addition to a rectal swab for microbiome testing, if
not already scheduled. Up to three bottles of doxycycline will be dispensed at the initial visit- a
sufficient quantity to ensure enough doxycycline for daily use until the next quarterly visit or the end
of study visit. If a control arm participant comes in to initiate doxycycline at a time that is before the
visit window for the next scheduled study visit, doxycycline will be dispensed and a rectal swab
obtained for microbiome evaluation. CBC and LFT evaluations for possible doxycycline toxicity will
occur at the month 3 and month 9 study visits per the SOE. If there is a delay in the participant’s
ability to come in for a doxycycline initiation visit, it is acceptable for doxycycline to be provided
through primary care or other provider in advance of visit. Roll over to doxycycline and the date of
starting doxycycline will be recorded on a CRF.

6.4 Evaluation at time of presumptive or diagnosed GC, CT and syphilis

6.4.1 Symptoms suggestive of STI for which presumptive STI treatment will be administered.
For those who have symptoms of an STI at a study visit given characteristic clinical presentation (such as
chancre, rash characteristic of syphilis, symptomatic urethritis or proctitis) such that the treating clinician will
provide empiric STI treatment while awaiting confirmatory testing, standard STI testing should be obtained from
urine/pharynx/rectum for GC/CT and blood for syphilis testing. In addition, the following specimens should be
obtained for resistance testing from symptomatic anatomic sites based on the clinical presentation as outlined
in Table 6.4.1.

NOTE: For specimens collected due to presumptive infection, the resistance testing specimens (swabs/urine
collection) should be held until definitive STI testing is available. If STI testing indicates no infection, the
specimens may be discarded. Alternatively, the site may send all specimens for storage. Specimens that are
not associated with a confirmed STI will be discarded when the data management team confirms absence of
STI. If there is uncertainty about whether STI if present, the specimens should be sent to the BioHUB for testing.

NOTE: Testing for evidence of resistance to GC, CT and syphilis should be conducted prior to STI treatment. If
collection of specimens before treatment is not possible, CT and syphilis testing can be conducted the same
day of treatment after treatment has been provided. Per the SURRG protocol, GC cultures should only be
collected before GC treatment.
 56

Table 6.4.1 Specimen collection for resistance testing at time of evaluation for STI and presumptive
STI treatment

Presumptive
infection Specimen collection
Urine Swab of GC culture per local
Buccal Rectal (Hologic chancre/ protocol (all anatomic
Urine
Swab Swab urine mucosal sites of sexual
(Zymo)
(Zymo) (Zymo) specimen lesion exposure in past 90
for M.gen) (Zymo) days)
Urethritis √ √ (urethral discharge)

Proctitis √ √
Syphilis with
chancre/mucosal √
legion

Syphilis diagnosis
without
√
chancre/mucosal
lesion
 57

6.4.2 Documented STI:

Individuals with positive GC and/or CT NAAT results or with serologic testing indicating a new syphilis infection
will be contacted to return for STI treatment as well as swabs of affected site for resistance testing or urine
collection, as outlined in Table 6.4.1 Specifically:
• Those with positive GC NAAT results will have
1) a swab obtained from the involved anatomic site and other sites of sexual contact in past 90
days (to include throat, rectum, urine) if indicated for GC culture-based phenotypic testing
according to local GC culture protocol, and
2) a swab/urine sample obtained from anatomic site of infection only for molecular resistance
testing.
• For those with CT infection, a swab or urine specimen will be collected (depending on the site of
infection) for molecular resistance testing.
• For those with confirmed rectal or pharyngeal CT, a swab will be collected for chlamydia cell culture.
Please see MOPS for specimen handing instructions.
• For those with syphilis serologies indicating new infection and in whom no chancres/mucosal lesions
are present, a buccal mucosal swab will be obtained for T. pallidum DNA amplification which, if
positive, will be used for resistance testing. If suspected syphilitic chancre is present, this should be
swabbed for molecular testing

Documented STI Specimen collection

GC culture per
Rectal or local protocol
Buccal Pharyngeal Rectal Urine Swab of (all anatomic
pharyngeal
Swab Swab Swab Collection chancre sites of sexual
swab
(Zymo) (Zymo) (Zymo) (Zymo) (Zymo) exposure in past
(Culture)
90 days
Chlamydia √* √* √* √*

Gonorrhea √* √* √* √
Syphilis with
chancre/mucosal √
legion
Secondary
syphilis (rash) √

*Specimen sent from site of confirmed infection only

6.5 Testing for resistance in setting of STI diagnoses on study

6.5.1 Testing for tetracycline resistance among N. gonorroheae

Positive GC cultures for all participants will be sent to either the University of Washington Neisseria Reference
laboratory for resistance testing led by our co-investigator, Dr Olusegun Soge at the UW/PHSKC STI program
or the SFDPH Public Health Laboratory for testing by Dr. Godfred Masinde. Both laboratories participate in the
Strengthening the US Response to Resistant Gonorrhea (SURRG) program. GC isolates collected in Seattle
and frozen isolates from San Francisco will be shipped to Dr. Soge on a monthly basis for agar dilution
antimicrobial susceptibility to a panel of antimicrobials including tetracycline, as part of the Antimicrobial
Resistance Lab Network (ARLN) supported by CDC. In addition, Dr. Soge’s laboratory will determine whether
tetracycline MICs increases among the subset with repeat GC infections during follow- up.
 58

6.5.2 Molecular testing for tetracycline resistance genes in C. trachomatis, T. pallidum and N. gonorrhoeae.
DNA from positive NAAT tests for C. trachomatis and N. gonorrhoeae and from buccal or chancre/mucosal lesion
swabs from those with newly diagnosed syphilis will be evaluated using established CRISPR/Cas9 techniques
to screen against a comprehensive library of antibiotic resistance mutationsin order to establish rapid throughput
methodology for the multicenter study. Tetracycline resistance will be assessed using FLASH (Finding Low
Abundance Sequences by Hybridization)) targeted sequencing of antimicrobial resistance genes that will then
be compared to an established antibiotic resistance database as well as evaluated for known tetracycline
mutations that have been documented in the target STIs or closely related organisms64. Molecular resistance
testing will conducted at the San Francisco Chan-Zuckerberg Biohub under the supervision of collaborating
investigator Dr. Langlier, who has extensive expertise in molecular diagnosis of mutations associated with
antibiotic resistance. Any TCN-resistance mutations in C. trachomatis or T. pallidum will be investigated further,
as clinically relevant tetracycline resistance has not been reported to date. The results will not be reported for use
in clinical care as these methods are experimental and have not been validated for clinical care.

6.6 Treatment of STIs

Local clinic standards will be followed for the treatment of STIs diagnosed by laboratory tests or when identified
syndromically. If doxycycline is used for treatment of an STI for an individual in the PEP arm, doxycycyline PEP
will be held during treatment and resumed when treatment completed.

6.7 Qualitative interviews

A qualitative interview will be offered to a subset of participants assigned to take doxycycline, approximately
20 per site, during months 3-12, and up to one month after study completion. Qualitative interviews will also
be offered to 10-20 providers at each of the sites A separate consent will be provided for these interviews.

6.8 Reenrollment of participants enrolled prior to 3/15/2020

Participants enrolled prior to the COVID pandemic will be re-enrolled starting 7/2020 with the same initial
assignment. At time of re-enrollment, these participants will undergo all day 0 evaluations with the exception of
the following
• Meningoccal vaccine history
• Medical & STI history
• Stool sample collection from first 50 PLWH and HIV uninfected on PrEP

7 ADVERSE EVENTS (AE) AND STUDY MONITORING

7.1 Adverse Event Collection Requirements

All AEs must be recorded on eCRFs if any of the following criteria have been met:
• All grade 2 and higher hematologic and hepatic laboratory abnormalities that are attributed to
doxycycline in the opinion of the site investigator
• All AEs meeting SAE definition
• All grade 3 and 4 adverse events judged to be associated with doxycycline (possibly,
probably, or definitely related)

Serious Adverse Events (SAEs)

An SAE is defined as any untoward medical occurrence that:
• Results in death
• Is life-threatening
• Requires inpatient hospitalization or prolongation of existing hospitalization
• Results in persistent or significant disability/incapacity
• Is an important medical event that may not be immediately life-threatening or result in death or
 59

hospitalization but may jeopardize the patient or may require intervention to prevent one of the
other outcomes listed in the definition above)

All AEs that are recorded must have their severity graded. To grade AEs, sites should refer to the Division
of AIDS Table for Grading the Severity of Adult and Pediatric Adverse Events (DAIDS AE Grading Table),
corrected Version 2.1, July 2017, which can be found on the DAIDS RSC website at https://rsc.tech-
res.com/docs/default-source/safety/division-of-aids-(daids)-table-for-grading-the-severity-of-adult-and-
pediatric-adverse-events-corrected-v-2-1.pdf

7.2 AE and SAE attribution to doxycycline

In those randomized to receive doxycycline PEP, all AEs and SAEs should have attribution recorded as
doxycycline-related or not doxycycline-related, in the judgment of the site investigator.

7.3 Study Monitoring

7.3.1 Study Team Monitoring

The study team will monitor the conduct of the study through monthly summary reports of arms of accrual, and
baseline characteristics and quarterly reports of data pooled over treatment arms of data completeness, specimen
collection, and AEs. The study will review individual participant-level safety data frequently to assess the relation
of all reported AEs to study treatment. On a monthly basis, the study team will review by- arm summaries of
premature study discontinuations and premature study treatment discontinuations (and reasons) and AEs.

7.3.2 Independent Monitor

Study conduct will be monitored by an independent monitor. Monitors will visit participating clinical research sites
to review the individual participant records, including consent forms, eCRFs, supporting data, laboratory specimen
records, and endpoints through laboratory and medical records (physicians’ progress notes, nurses’ notes,
individuals’ hospital charts), to ensure protection of study participants, compliance with the protocol, and
accuracy and completeness of records. The monitors also will inspect sites’ regulatory files to ensure that
regulatory requirements are being followed and sites’ pharmacies to review product storage and management.

7.3.3 Data safety and monitoring board (DSMB)

An independent data safety and monitoring board (DSMB) will be convened for this study with expertise in STIs,
PrEP, and HIV care and a biostatistician. The purpose of the DSMB is to monitor the study for operational futility,
social harms, and efficacy. The DSMB will evaluate the progress of the project, including periodic assessments
of accrual, retention, safety, performance and variation of the project sites, and other factors that can affect
project implementation. The DSMB will review the study after 1/2 of follow- up time with pre-specified stopping
rules in terms of the efficacy of doxycycline PEP in reducing the incidence of gonorrhea, chlamydia, and syphilis
overall, and the ability of the study to meet its objectives. The DSMB will review STI endpoints which have been
adjudicated by the STI Endpoints Committee. The DMSB will consider factors external to the project when
relevant information becomes available, such as policy changes or scientific developments that may have an
impact on project implementation, safety, and integration of STI PEP in the STI and HIV care clinics.

The DSMB will conduct reviews every six month and convene by teleconference. Open reports containing
accrual and retention rates, participant characteristics, serious adverse events, and social, will be sent to the
protocol team and DSMB members the week prior to the DSMB meeting. Only the DSMB members and the
unblinded biostatistician will receive password-protected closed reports of STI endpoints by randomization arm.
 60

8 CLINICAL MANAGEMENT ISSUES

8.1 Toxicity
Only toxicities related to study medications provided through the study will be considered in the toxicity
management section.

Grade 1 or 2
Participants who develop Grade 1 or 2 toxicity (per DAIDS toxicity table) felt to be related to
doxycycyline may continue study treatment at the discretion of the site investigator with close follow-
up. If a participant chooses to discontinue study treatment, the site should notify the study
protocol team within 7 days.These participants will remain on study, off study treatment and have all
evaluations performed per the SOE.

Grade 3
• Participants who develop a Grade 3 toxicity thought by the site investigator to be related to study
drug should have doxycycline PEP withheld and the site should consult with the core protocol
team. The participant should be reevaluated weekly until the AE returns to Grade ≤2, at which
time study drug may be reintroduced at the discretion of the site investigator in consultation with
the protocol team.
• Participants experiencing Grade 3 toxicity requiring permanent discontinuation of doxycycline
PEP should be followed weekly until resolution of the toxicity. Participants will have premature
study treatment discontinuation evaluations performed as noted on the SOE. These participants
will remain on study, off study treatment and have all evaluations performed per the SOE.

Grade 4
• Participants with Grade 4 asymptomatic laboratory abnormalities may continue doxycycline PEP
if the site investigator has compelling evidence that the toxicity is NOT related to study drug.
• Participants who develop a Grade 4 symptomatic toxicity will have doxycycline PEP
permanently discontinued and the site should notify study team within 72 hours.
• Participants experiencing Grade 4 toxicity requiring permanent discontinuation of doxycycline
PEP should be followed weekly until resolution of the AE or return to baseline. These
participants will remain on study, off study treatment and have all evaluations performed per the
SOE.
•
8.2 Specific Management of Toxicities Related to Study-Provided Drugs
Possible intracranial hypertension (IH)
Participants taking doxycycline who report new or worsening headaches, visual changes, or vision loss should
have doxycycline temporarily discontinued and evaluated for IH if these symptoms persist, including a
fundoscopic exam to look for papilledema. If IH is ruled-out or an alternate etiology identified, doxycycline may
be restarted, at the discretion of the site investigator.

Skin erythema
Increased photosensitivity is a known possible side effect of doxycycline. Doxycycline may be discontinued if
skin erythema develops at the discretion of the site investigator and may reinstituted when resolved at the
discretion of the site investigation. Any serious skin reaction judged by the local investigator to be related to
doxycycline should lead to permanent discontinuation of doxycycline .

Fixed drug eruption

Suspected fixed drug eruption should be evaluated for possible etiologies – if a doxycycline fixed drug eruption
is suspected, doxycycline should be stopped.

Allergic reactions
 61

Doxycycline should be discontinued permanently if a serious allergic reaction is suspected. These participants
will remain on study, off study treatment and have all evaluations performed per the SOE.

9 CRITERIA FOR DISCONTINUATION

9.1 Premature Study Treatment Discontinuation

• Requirement for prohibited concomitant medications or other contraindication to doxycycline

• Occurrence of an adverse event requiring discontinuation of doxycycline
• Request by participant to terminate study treatment
• Clinical reasons believed life threatening by the physician, even if not addressed in the toxicity
section of the protocol
• Requirement for chronic tetracycline use ( >14 days)

Participants who stop doxycycline PEP should be continued on study, off doxycycline PEP with
continued evaluations as per the SOE. The reason for doxycycline discontinuation should be
recorded.

9.2 Premature Study Discontinuation

• Request by the participant to withdraw

• Request of the primary care provider if she or he thinks the study is no longer in the best
interest of the participant
• Participant judged by the investigator to be at significant risk of failing to comply with the
provisions of the protocol as to cause harm to self or seriously interfere with the validity of the
study results
• At the discretion of the IRB/EC, NIAID, Office for Human Research Protections (OHRP), other
government agencies as part of their duties, investigator, or industry supporter

10 STATISTICAL CONSIDERATIONS

10.1 Outcome measures

10.1.1 Primary outcome:

Combined incidence of GC, CT, or early syphilis infection by laboratory- based diagnosis (e.g., positive GC or CT
based on NAAT, syphilis based on four-fold increase in non- treponemal titers or positive darkfield on exudate
from a lesion). Incident STI diagnoses will be ascertained through the study through periodic testing, interim visits
for STI symptoms, as well as STI surveillance from the San Francisco and King County Departments of Health
and by medical records provided for participants who receive their care from other clinical sites.

10.1.2 Secondary outcomes:

• Safety of doxycycline PEP, measured by evaluation of all Grade 3 and 4 AEs attributed to study medication.
• Tolerability, measured using a brief quarterly questionnaire administered to both intervention and control groups
evaluating side effects commonly associated with doxycycline as well as recording any discontinuations of
doxycycline by participants and their reasons.
• Acceptability, assessed through a brief questionnaire for all PEP recipients administered at month 6 and 12 and
in-depth structured qualitative interviews for a subset, conducted within 6 months of month 12 visit. A separate
consent will be provided for the in-depth qualitative interview at the time of this interview.
• Doxycycline coverage of sexual contacts in the prior 3 months, ascertained by sexual behavior and doxycycline
 62

use from the web-based app, augmented by quarterly adherence questionnaires and pill counts.
• Longitudinal exposure to doxcycyline will be assessed using hair collection, with a semiquantitive evaluation in
the control arm (doxycycline present vs. absent) and in the PEP arm (doxycycline concentration high vs. low).
• Adherence to HIV medications as reflected in viral suppression among participants living with HIV, assessed
by chart review of HIV RNA levels conducted as standard clinical care.
• Discontinuation of PrEP, based on self-report
• Residual hair samples of participants without HIV infection on PrEP will be archived for future tenofovir testing
to measure adherence to Truvada PrEP.

10.2 Statistical power and analysis

Primary endpoint: We selected a sample size for each cohort (MSM/TGW living with HIV and MSM/TGW on
PrEP) to achieve 80% power with 0.05 two-sided Type 1 error, for the endpoint of any lab-detected incident early
syphilis, GC and/or CT infection, based on quarterly assessments for each person, 10% annual loss to follow-up
and an intra-class correlation of 0.2, reflecting the high prevalence of STI re-infection that has been observed in
these populations. With these assumptions, a cohort of approximately 390 of MSM/TGW living with HIV and 390
of MSM/TGW on PrEP (130 in the control and 260 in the intervention for each cohort) will provide 80% power to
detect a decrease in quarterly STI prevalence from 10% to 5%, which corresponds to an annual reduction in
combined STI incidence from 34% to 19% (Table 10.2.1). With allowance for 10% lost to follow-up, the sample
size for each cohort will be approximately 390 participants, for a total sample size of 780. Based on 2017 STI
incidence data from San Francisco and Seattle PrEP cohorts for both GC and CT, we expect in the control arm in
each quarter approximately 5% of participants to have GC, 5% CT and 2% early syphilis, acknowledging some
will be diagnosed with more than one infection. Based on the higher susceptibility of GC to TCN in the US vs
France (75% vs < 50%11, 20) and the 70% reduction of CT and syphilis with PEP in IPERGAY,1 we anticipate
effectiveness of doxycycline PEP to be 35% for GC, 65% for CT and 65% for syphilis. Given these assumptions,
with a 2:1 randomization we expect to see 61 vs 47 GC infections, 47 vs 33 CT infections and 19 vs 13 syphilis
infections in the doxycycline PEP and control arms respectively, some of which will occur in the same individuals.
Sample sizes were computed using nQuery for repeated measures for two proportions. The analysis will compare
time-averaged proportion of infections in each quarter between arms, using repeated measure methods.

Table 10.2.1: Power Table for N = 390; 2:1 randomization doxycycline PEP: SOC. Sample size
assumes 370 fully evaluable participants.

Intervention arm Power to detect 50% reduction Power to detect 65% reduction
Control arm quarterly
quarterly
prevalence
prevalence ICC = 0.2 ICC = 0.1 ICC = 0.2 ICC = 0.1

10% 5% 80% 87% 96% 99%

7.5% 3.75% 68% 76% 90% 95%

5% 2.5% 51% 59% 77% 85%

3% 1.5% 35% 41% 57% 65%

2% 1% 26% 30% 43% 50%

 63

Within the total enrollment of 780, we will not require the enrollment of HIV-infected and uninfected participants
to be equal. Anticipating the possibility of lower enrollment but possibility of equivalent or higher STI rates in the
HIV-infected cohort, Table 10.2.2 assesses the power characteristics of the study with a cohort that contributes
from 30% to 50% of the total enrolled, with quarterly events rates of 10% and 12%. With a higher event rate or
a lower intraclass correlation, the smaller cohort retains reasonable power to detect a 50% reduction.

Table 10.2.2: Power characteristics for reduced sample size in a cohort, assuming event rate of 10%
and 12% STIs occurring each quarter in SOC arm

Total Evaluable ICC Doxy: SOC Power to detect Power to detect

sample size in cohort 50% reduction 50% reduction
(10% in SOC) (12% in SOC)
370 (50% of total) 0.1 246:123 87% 92%
370 0.2 246:123 80% 86%
296 (40% of total) 0.1 196:98 79% 86%
296 0.2 196:98 71% 78%
259 (35% of total) 0.1 174:87 74% 82%
259 0.2 174:87 66% 74%
222 (30% of total) 0.1 148:74 68% 76%
222 0.2 148:74 60% 67%

Secondary endpoints include evaluation of PEP effectiveness on incidence of each specific STI and on time to
first STI, as well as by site of STI infection (oral, rectal, urethral). Impact of risk practices and sexual activity on STI
incidence will be compared between arms. Survey data will be analyzed to understand factors impacting
acceptability of doxycycline PEP, including demographic factors, socioeconomic status, substance use, history
of STIs, HIV infection status, and experience with PEP. The qualitative interviews will be coded using inductive
(i.e., codes arise from careful reading of the text) and deductive (i.e., codes arise a priori from topics covered by
the interview guide) approaches, which when employed together have proven useful in health services research.
Interviews will be coded by at least two research team members who will practice on a selection of transcripts
until >90% agreement is reached. Dedoose, a web-based program, will be used to facilitate indexing, coding, and
searching. The qualitative team will meet regularly to group codes into categories and develop major and minor
themes.

For resistance we will assess the proportion with tetracycline resistance in visits with N. gonorrheae and S.
aureus. Exploratory endpoints include incidence of tetracycline resistance in C. trachomatis and T. pallidum
using molecular techniques (e.g., CRISPR and CAS-9), for which the molecular technologies are new and the
clinical significance of resistance mutations is less clear. Stool samples from 50 participants living with HIV and
50 participants on PrEP will be obtained at baseline, 6 and 12 months for metagenomic evaluation targeting TCN
genes. Overall TCN resistance gene abundance (expressed as dpM/patient) and the number of distinct TCN
resistance genes present per participant will be assessed at baseline and evaluated for change over time during
doxycycline use. Rectal swabs will be collected and stored for future evaluation of shifts in the fecal microbiome,
decrease in flora diversity, and quantification of TCN-resistant abundance and diversity

10.3 Endpoint adjudication

All STI endpoints will be reviewed by a blinded, independent Endpoint Adjudication Committee, following CDC
guidelines for the diagnosis of STIs.52 Incidence of individual STIs and tetracycline resistance will also be
evaluated.
 64

10.4 Post COVID follow-up

The team will monitor discontinuation rates in the participants enrolled prior to the COVID epidemic (3/15/2020).
If there is a differential discontinuation in the active vs. control arm, this will be discussed with DSMB and the
team statistician with consideration of additional enrollments to ensure adequate power for the primary endpoint.

10.5 Analyses after 5/13/2022 closure to enrollment

Analyses after 5/13/2022 closure to enrollment and doxycycline provision to control will include the following:

STI incidence:
- Comparison of STI incidence per quarter in control arm before and after doxycycline initiation
- Comparison of STI incidence per quarter in all participants receiving doxycycline (including those in
control arm after rolled over to receive doxycycline) compared to incidence on control arm prior to
doxycycline roll over

Antimicrobial resistance
- Evaluation of tetracycline resistance in Staphylococcus aureus, commensal Neisseria in comparison
to the control arm and to individual baseline
- Evaluation of genotypic or phenotypic tetracycline resistance in bacterial STIs in all participants who
receive doxycycline compared to control arm participants, prior to receipt of doxycycline.
- Evaluation of changes in the gut microbiome and total gut burden of tetracycline resistance before and
after taking doxycycline PEP

Adherence
- Evaluation of doxycycline coverage of qualifying sex before and after study closure 5/13/2022

Acceptability
- Proportion of control arm participants who opt to take study provided doxycycline. Control arm
participants who terminate study participation will have the reason for termination recorded to
determine if stopping to receive doxycycline outside of the study.

11 PARTICIPANTS

11.1 Institutional Review Board (IRB) Review

The study protocol, site-specific informed consent forms, participant education and recruitment materials, and
other requested documents—including any subsequent modifications—will be reviewed and approved by the
UCSF IRB, as the single IRB of record responsible for oversight of research conducted at the study sites.
Subsequent to initial review and approval, the UCSF IRB will review the study at least annually.

11.2 Informed Consent

A signed consent form will be obtained from the participant.The consent form will describe the purpose of the
study, the procedures to be followed, and the risks and benefits of participation. A copy of the consent form will
be given to the participant and this fact will be documented in the participant’s record. Electronic consenting will
be an option to reduce face to face exposure during the COVID epidemic.

11.3 Study records

Each study site will establish a standard operating procedure for confidentiality protection. Each site will ensure
that study records including administrative documentation and regulatory documentation as well as
documentation related to each participant enrolled in the study, including informed consent forms, locator forms,
case report forms, notations of all contacts with the participant, and all other source documents are stored in a
secure manner.
 65

11.4 Confidentiality
Participants’ study information will not be released without their written permission, except as necessary for
oversight by:
- The Protocol co-Chairs or designees
- Study funders
- University of California, San Francisco IRB
- University of Washington IRB

All laboratory specimens, evaluation forms, reports, and other records that leave the site will be identified by
coded number only to maintain participant confidentiality. The exceptions are STI testing results which are
subject to local and state reporting which is names-based. Local public health may contact participants
diagnosed with STIs for the purpose of surveillance and partner notification. Participants will be informed prior
to STI testing that results are reportable and may lead to contact by local public health if results are positive for
infection. In addition, GC culture specimens will be evaluated for drug resistance as part of an existing GC
program evaluating clinical isolates and may contain patient identifiers such as MRN, name or date of visit,
according to local laboratory requirements and practice.

All records will be kept locked. All computer entry and networking programs will be done with coded numbers only.
Clinical information will not be released without written permission of the participant, except as necessary for
monitoring by the IRB/EC, NIAID, OHRP, other local, US, and international regulatory entities as part of their
duties, or the industry supporters or designees.

12 BIOHAZARD CONTAINMENT

As the transmission of HIV and other blood-borne pathogens can occur through contact with contaminated
needles, blood, and blood products, appropriate blood and secretion precautions will be employed by all
personnel in the drawing of blood and shipping and handling of all specimens for this study, as currently
recommended by the CDC and the National Institutes of Health.

All dangerous goods and materials, including diagnostic specimens and infectious substances, must be
transported using packaging mandated by CFR 42 Part 72. Please refer to instructions detailed in the
International Air Transport Association (IATA) Dangerous Goods Regulations.
 66

REFERENCES

1. Molina, J.M., I. Charreau, C. Chidiac, G. Pialoux, E. Cua, C. Delaugerre, C. Capitant, D. Rojas-

Castro, J. Fonsart, B. Bercot, C. Bebear, L. Cotte, O. Robineau, F. Raffi, P. Charbonneau, A. Aslan,
J. Chas, L. Niedbalski, B. Spire, L. Sagaon-Teyssier, D. Carette, S.L. Mestre, V. Dore, and L.
Meyer, Post-exposure prophylaxis with doxycycline to prevent sexually transmitted infections in
men who have sex with men: an open-label randomised substudy of the ANRS IPERGAY trial.
Lancet Infect Dis, 2017.

2. CDC, Sexually Transmitted Disease Surveillance 2016. 2017.

3. Volk, J.E., J.L. Marcus, T. Phengrasamy, D. Blechinger, D.P. Nguyen, S. Follansbee, and C.B.
Hare, No New HIV Infections With Increasing Use of HIV Preexposure Prophylaxis in a Clinical
Practice Setting. Clin Infect Dis, 2015. 61(10): p. 1601-3.

4. Montano, M.A., J.C. Dombroski, L.A. Barbee, M.R. Golden, and C.M. Khosropour. Changes in
sexual behavior and STI diagnoses among MSM using PrEP in Seattle, WA (Abstract #979). in
Conference on Retroviruses and Opportunistic Infections (CROI). 2018. Boston, MA.

5. Oliver, S.E., M. Aubin, L. Atwell, J. Matthias, A. Cope, V. Mobley, A. Goode, S. Minnerly, J. Stoltey,
H.M. Bauer, R.R. Hennessy, D. DiOrio, R.N. Fanfair, T.A. Peterman, and L. Markowitz, Ocular
Syphilis - Eight Jurisdictions, United States, 2014-2015. MMWR Morb Mortal Wkly Rep, 2016.
65(43): p. 1185-1188.

6. Bowen, V., J. Su, E. Torrone, S. Kidd, and H. Weinstock, Increase in incidence of congenital
syphilis - United States, 2012-2014. MMWR Morb Mortal Wkly Rep, 2015. 64(44): p. 1241-5.

7. Cunningham, S.D., G. Olthoff, P. Burnett, A.M. Rompalo, and J.M. Ellen, Evidence of heterosexual
bridging among syphilis‐positive men who have sex with men. Sexually Transmitted Infections,
2006. 82(6): p. 444-445.

8. Wi, T., M.M. Lahra, F. Ndowa, M. Bala, J.-A.R. Dillon, P. Ramon-Pardo, S.R. Eremin, G. Bolan, and
M. Unemo, Antimicrobial resistance in Neisseria gonorrhoeae: Global surveillance and a call for
international collaborative action. PLOS Medicine, 2017. 14(7): p. e1002344.

9. Gianecini, R., C. Oviedo, G. Stafforini, and P. Galarza, Neisseria gonorrhoeae Resistant to

Ceftriaxone and Cefixime, Argentina. Emerging Infectious Diseases, 2016. 22(6): p. 1139-1141.

10. Camara, J., J. Serra, J. Ayats, T. Bastida, D. Carnicer-Pont, A. Andreu, and C. Ardanuy, Molecular
characterization of two high-level ceftriaxone-resistant Neisseria gonorrhoeae isolates detected in
Catalonia, Spain. J Antimicrob Chemother, 2012. 67(8): p. 1858-60.

11. Kirkcaldy, R.D., A. Harvey, J.R. Papp, C. Del Rio, O.O. Soge, K.K. Holmes, E.W. Hook, 3rd, G.
Kubin, S. Riedel, J. Zenilman, K. Pettus, T. Sanders, S. Sharpe, and E. Torrone, Neisseria
gonorrhoeae Antimicrobial Susceptibility Surveillance - The Gonococcal Isolate Surveillance
Project, 27 Sites, United States, 2014. MMWR Surveill Summ, 2016. 65(7): p. 1-19.

12. BBC. 'World's worst' super-gonorrhoea man cured. 2018 4/26/2018]; Available from:
http://www.bbc.com/news/health-43840505.
 67

13. Lahra, M.M., N. Ryder, and D.M. Whiley, A new multidrug-resistant strain of Neisseria gonorrhoeae
in Australia. N Engl J Med, 2014. 371(19): p. 1850-1.

14. Cohen, M.S., I.F. Hoffman, R.A. Royce, P. Kazembe, J.R. Dyer, C.C. Daly, D. Zimba, P.L.
Vernazza, M. Maida, S.A. Fiscus, and J.J. Eron, Jr., Reduction of concentration of HIV-1 in semen
after treatment of urethritis: implications for prevention of sexual transmission of HIV-1. AIDSCAP
Malawi Research Group. Lancet, 1997. 349(9069): p. 1868-73.

15. Schacker, T., A.J. Ryncarz, J. Goddard, K. Diem, M. Shaughnessy, and L. Corey, Frequent
recovery of HIV-1 from genital herpes simplex virus lesions in HIV-1-infected men. JAMA, 1998.
280(1): p. 61-6.

16. CDC. Understanding the HIV Care Continuum. 2017; Available from:
https://www.cdc.gov/hiv/pdf/library/factsheets/cdc-hiv-care-continuum.pdf.

17. The Lancet, H., U=U taking off in 2017. Lancet HIV, 2017. 4(11): p. e475.

18. Harrison, W.O., R.R. Hooper, P.J. Wiesner, A.F. Campbell, W.W. Karney, G.H. Reynolds, O.G.
Jones, and K.K. Holmes, A trial of minocycline given after exposure to prevent gonorrhea. N Engl J
Med, 1979. 300(19): p. 1074-8.

19. Steen, R., M. Chersich, A. Gerbase, G. Neilsen, A. Wendland, F. Ndowa, E.A. Akl, Y.R. Lo, and
S.J. de Vlas, Periodic presumptive treatment of curable sexually transmitted infections among sex
workers: a systematic review. Aids, 2012. 26(4): p. 437-45.

20. La Ruche, G., A. Goubard, B. Bercot, E. Cambau, C. Semaille, and P. Sednaoui, Gonococcal
infections and emergence of gonococcal decreased susceptibility to cephalosporins in France, 2001
to 2012. Euro Surveill, 2014. 19(34).

21. Molina, J.M., I. Charreau, C. Chidiac, G. Pialoux, E. Cua, C. Delaugerre, C. Capitant, D. Rojas-
Castro, J. Fonsart, B. Bercot, C. Bebear, L. Cotte, O. Robineau, F. Raffi, P. Charbonneau, A. Aslan,
J. Chas, L. Niedbalski, B. Spire, L. Sagaon-Teyssier, D. Carette, S.L. Mestre, V. Dore, and L.
Meyer. Supplement to: Molina JM, Charreau I, Chidiac C, et al. Post-exposure prophylaxis with
doxycycline to prevent sexually transmitted infections in men who have sex with men: an open-label
randomised substudy of the ANRS IPERGAY trial. Lancet Infect Dis 2017. 2017; Available from:
https://www.sciencedirect.com/science/article/pii/S1473309917307259?via%3Dihub#sec1.

22. Bolan, R.K., M.R. Beymer, R.E. Weiss, R.P. Flynn, A.A. Leibowitz, and J.D. Klausner, Doxycycline
prophylaxis to reduce incident syphilis among HIV-infected men who have sex with men who
continue to engage in high-risk sex: a randomized, controlled pilot study. Sex Transm Dis, 2015.
42(2): p. 98-103.

23. Gaillard, T., M. Madamet, and B. Pradines, Tetracyclines in malaria. Malar J, 2015. 14: p. 445.

24. van Zuuren, E.J. and Z. Fedorowicz, Interventions for rosacea: abridged updated Cochrane
systematic review including GRADE assessments. Br J Dermatol, 2015. 173(3): p. 651-62.

25. Bienenfeld, A., A.R. Nagler, and S.J. Orlow, Oral Antibacterial Therapy for Acne Vulgaris: An
Evidence-Based Review. Am J Clin Dermatol, 2017. 18(4): p. 469-490.

26. Sloan, B. and N. Scheinfeld, The use and safety of doxycycline hyclate and other second-
generation tetracyclines. Expert Opin Drug Saf, 2008. 7(5): p. 571-7.
 68

27. Turner, R.B., C.B. Smith, J.L. Martello, and D. Slain, Role of doxycycline in Clostridium difficile
infection acquisition. Ann Pharmacother, 2014. 48(6): p. 772-6.

28. Tariq, R., J. Cho, S. Kapoor, R. Orenstein, S. Singh, D.S. Pardi, and S. Khanna, Low Risk of
Primary Clostridium difficile Infection With Tetracyclines: A Systematic Review and Metaanalysis.
Clin Infect Dis, 2018. 66(4): p. 514-522.

29. Doernberg, S.B., L.G. Winston, D.H. Deck, and H.F. Chambers, Does doxycycline protect against
development of Clostridium difficile infection? Clin Infect Dis, 2012. 55(5): p. 615-20.

30. Khanna, S., Do tetracyclines have the potential to reduce the risk of Clostridium difficile infection?
Expert Review of Anti-infective Therapy, 2018: p. 1-3.

31. Magnuson, H.J., H. Eagle, and R. Fleischman, The minimal infectious inoculum of Spirochaeta
pallida (Nichols strain) and a consideration of its rate of multiplication in vivo. Am J Syph Gonorrhea
Vener Dis, 1948. 32(1): p. 1-18.

32. Cumberland, M.C. and T.B. Turner, The rate of multiplication of Treponema pallidum in normal and
immune rabbits. Am J Syph Gonorrhea Vener Dis, 1949. 33(3): p. 201-12.

33. Lafond, R.E. and S.A. Lukehart, Biological basis for syphilis. Clin Microbiol Rev, 2006. 19(1): p. 29-
49.

34. Nadelman, R.B., J. Nowakowski, D. Fish, R.C. Falco, K. Freeman, D. McKenna, P. Welch, R.
Marcus, M.E. Aguero-Rosenfeld, D.T. Dennis, and G.P. Wormser, Prophylaxis with single-dose
doxycycline for the prevention of Lyme disease after an Ixodes scapularis tick bite. N Engl J Med,
2001. 345(2): p. 79-84.

35. Levett, P.N., Leptospirosis. Clin Microbiol Rev, 2001. 14(2): p. 296-326.

36. Schneider, M.C., J. Velasco-Hernandez, K.D. Min, D.G. Leonel, D. Baca-Carrasco, M.E. Gompper,
R. Hartskeerl, and C. Munoz-Zanzi, The Use of Chemoprophylaxis after Floods to Reduce the
Occurrence and Impact of Leptospirosis Outbreaks. Int J Environ Res Public Health, 2017. 14(6).

37. Burkot, T.R., J. Piesman, and R.A. Wirtz, Quantitation of the Borrelia burgdorferi outer surface
protein A in Ixodes scapularis: fluctuations during the tick life cycle, doubling times, and loss while
feeding. J Infect Dis, 1994. 170(4): p. 883-9.

38. Wanninger, S., M. Donati, A. Di Francesco, M. Hässig, K. Hoffmann, H.M.B. Seth-Smith, H. Marti,
and N. Borel, Selective Pressure Promotes Tetracycline Resistance of Chlamydia Suis in Fattening
Pigs. PLOS ONE, 2016. 11(11): p. e0166917.

39. Dugan, J., D.D. Rockey, L. Jones, and A.A. Andersen, Tetracycline resistance in Chlamydia suis
mediated by genomic islands inserted into the chlamydial inv-like gene. Antimicrob Agents
Chemother, 2004. 48(10): p. 3989-95.

40. Mirajkar, N.S., P.R. Davies, and C.J. Gebhart, Antimicrobial Susceptibility Patterns of Brachyspira
Species Isolated from Swine Herds in the United States. J Clin Microbiol, 2016. 54(8): p. 2109-19.

41. Centurion-Lara, A., C. Castro, W.C. van Voorhis, and S.A. Lukehart, Two 16S-23S ribosomal DNA
intergenic regions in different Treponema pallidum subspecies contain tRNA genes. FEMS
Microbiol Lett, 1996. 143(2-3): p. 235-40.
 69

42. Mitchell, S.J., J. Engelman, C.K. Kent, S.A. Lukehart, C. Godornes, and J.D. Klausner,
Azithromycin-resistant syphilis infection: San Francisco, California, 2000-2004. Clin Infect Dis,
2006. 42(3): p. 337-45.

43. Stamm, L.V., Global challenge of antibiotic-resistant Treponema pallidum. Antimicrob Agents
Chemother, 2010. 54(2): p. 583-9.

44. Lucet, J.-C. and B. Regnier, Screening and Decolonization: Does Methicillin-Susceptible
Staphylococcus aureus Hold Lessons for Methicillin-Resistant S. aureus? Clinical Infectious
Diseases, 2010. 51(5): p. 585-590.

45. Stevens, D.L., A.L. Bisno, H.F. Chambers, E.P. Dellinger, E.J. Goldstein, S.L. Gorbach, J.V.
Hirschmann, S.L. Kaplan, J.G. Montoya, J.C. Wade, and A. Infectious Diseases Society of, Practice
guidelines for the diagnosis and management of skin and soft tissue infections: 2014 update by the
Infectious Diseases Society of America. Clin Infect Dis, 2014. 59(2): p. e10-52.

46. Jakobsson, H.E., C. Jernberg, A.F. Andersson, M. Sjölund-Karlsson, J.K. Jansson, and L.
Engstrand, Short-Term Antibiotic Treatment Has Differing Long-Term Impacts on the Human Throat
and Gut Microbiome. PLOS ONE, 2010. 5(3): p. e9836.

47. Shanahan, F., The colonic microbiota in health and disease. Curr Opin Gastroenterol, 2013. 29(1):
p. 49-54.

48. Blaser, M.J., The Past and Future Biology of the Human Microbiome in an Age of Extinctions. Cell,
2018. 172(6): p. 1173-1177.

49. Petousis-Harris, H., J. Paynter, J. Morgan, P. Saxton, B. McArdle, F. Goodyear-Smith, and S.

Black, Effectiveness of a group B outer membrane vesicle meningococcal vaccine against
gonorrhoea in New Zealand: a retrospective case-control study. Lancet, 2017. 390(10102): p. 1603-
1610.

50. ACIP. Recommended Immunization Schedule for Adults Aged 19 Years or Older, United States,
2018. 2018; Available from: https://www.cdc.gov/vaccines/schedules/hcp/imz/adult.html#f10.

51. Taylor-Robinson, D. and J.S. Jensen, Mycoplasma genitalium: from Chrysalis to multicolored
butterfly. Clin Microbiol Rev, 2011. 24(3): p. 498-514.

52. Workowski, K.A., G.A. Bolan, C. Centers for Disease, and Prevention, Sexually transmitted
diseases treatment guidelines, 2015. MMWR Recomm Rep, 2015. 64(RR-03): p. 1-137.

53. Mena, L.A., T.F. Mroczkowski, M. Nsuami, and D.H. Martin, A randomized comparison of
azithromycin and doxycycline for the treatment of Mycoplasma genitalium-positive urethritis in men.
Clin Infect Dis, 2009. 48(12): p. 1649-54.

54. Schwebke, J.R., A. Rompalo, S. Taylor, A.C. Sena, D.H. Martin, L.M. Lopez, S. Lensing, and J.Y.
Lee, Re-evaluating the treatment of nongonococcal urethritis: emphasizing emerging pathogens--a
randomized clinical trial. Clin Infect Dis, 2011. 52(2): p. 163-70.

55. Cho, I., S. Yamanishi, L. Cox, B.A. Methe, J. Zavadil, K. Li, Z. Gao, D. Mahana, K. Raju, I. Teitler,
H. Li, A.V. Alekseyenko, and M.J. Blaser, Antibiotics in early life alter the murine colonic
microbiome and adiposity. Nature, 2012. 488(7413): p. 621-6.
 70

56. Angelakis, E., M. Million, S. Kankoe, J.C. Lagier, F. Armougom, R. Giorgi, and D. Raoult, Abnormal
weight gain and gut microbiota modifications are side effects of long-term doxycycline and
hydroxychloroquine treatment. Antimicrob Agents Chemother, 2014. 58(6): p. 3342-7.

57. Lane, J.A., L.J. Murray, I.M. Harvey, J.L. Donovan, P. Nair, and R.F. Harvey, Randomised clinical
trial: Helicobacter pylori eradication is associated with a significantly increased body mass index in
a placebo-controlled study. Aliment Pharmacol Ther, 2011. 33(8): p. 922-9.

58. Kamada, T., J. Hata, H. Kusunoki, M. Ito, S. Tanaka, Y. Kawamura, K. Chayama, and K. Haruma,
Eradication of Helicobacter pylori increases the incidence of hyperlipidaemia and obesity in peptic
ulcer patients. Dig Liver Dis, 2005. 37(1): p. 39-43.

59. Angelakis, E., N. Armstrong, C. Nappez, M. Richez, E. Chabriere, and D. Raoult, Doxycycline
assay hair samples for testing long-term compliance treatment. Journal of Infection, 2015. 71(5): p.
511-517.

60. Geisler, W.M., W.D. Koltun, N. Abdelsayed, J. Burigo, L. Mena, S.N. Taylor, B.E. Batteiger, A.R.
Thurman, I.I.I.E.W. Hook, T.A. Vaughn, M.P. Annett, R.A. Muenzen, and J. Caminis, Safety and
Efficacy of WC2031 Versus Vibramycin for the Treatment of Uncomplicated Urogenital Chlamydia
trachomatis Infection: A Randomized, Double-blind, Double-Dummy, Active-Controlled, Multicenter
Trial. Clinical Infectious Diseases, 2012. 55(1): p. 82-88.

61. FDA. 505(b)(2) Efficacy Supplement, Resubmission/Class 2 Doxycycline Hyclate DR 4/1/2019];

Available from: https://www.fda.gov/media/86171/download.

62. Lexicomp, Doxycycline (Lexi-Drugs) 2019.

63. Mayne. Doxycycline Prescribing information 1/5/2019]; Available from:

https://dailymed.nlm.nih.gov/dailymed/getFile.cfm?setid=54ce5887-5e72-4d7a-a821-
936dd3ffa68c&type=pdf&name=54ce5887-5e72-4d7a-a821-936dd3ffa68c

64. Jia, B., A.R. Raphenya, B. Alcock, N. Waglechner, P. Guo, K.K. Tsang, B.A. Lago, B.M. Dave, S.
Pereira, A.N. Sharma, S. Doshi, M. Courtot, R. Lo, L.E. Williams, J.G. Frye, T. Elsayegh, D. Sardar,
E.L. Westman, A.C. Pawlowski, T.A. Johnson, F.S. Brinkman, G.D. Wright, and A.G. McArthur,
CARD 2017: expansion and model-centric curation of the comprehensive antibiotic resistance
database. Nucleic Acids Res, 2017. 45(D1): p. D566-D573.
 71

Doxy PEP Protocol Summary of Changes

Protocol
date/version Changes made
number
7/17/19 1. Added TGW to all sections of the protocol
version 2.0 2. Included sentence: “Condomless sexual contact is defined as receptive
anal, insertive anal, vaginal, or oral sex (which includes oral/anal and
oral/genital contact) or use of shared sex toys without a condom used for
the entire time.”
3. Section 6.3.10 includes “STI” before risk reduction and HIV
“prevention”…and “local standard of care”
4. Added “ An additional reimbursement will be provided for participants who
consent to the optional stool sample collection at the baseline, 6 month
and 12 month timepoints.”
10/9/19 1. Included syphilis testing and rectal swabs stored for future microbiome
version 3.0 testing to the procedural table
2. Added section 6.2.4 “ Termination visit: In the event that a participant
prematurely discontinues study participation, the evaluations for the
termination visit shoud be completed if possible.
3. Added CD4 cell count to section 6.3.20
4. Note for section 6.4.1 NOTE: For specimens collected due to presumptive
infection, the resistance testing sepcimens (swabs/urine collection)
should be held until definitive STI testing is available. If STI testing
indicates no infection, the specimens may be discarded. If there is
uncertainty about whether STI if present, the specimens should be sent to
the BioHUB for testing”- Also added syphilis diagnosis without chancre to
table
5. Sentence added to section 10.1.2. bullet point three “conducted within 6
months of month 12 visit. A separate consent will be provided for the in-
depth qualitative interview at the time of this interview.”
1/10/2020 1. Added Vivek Jain and Emma Bainbridge to the study team
version 4.0 2. Included the following sentence to the exclusion criteria in section 4.2.2:
“Current use of warfarin, as intermittent doxycycline use can lead to an
unpredictable impact on INR”
3. Added section 6.3.21 “ Serum for storage (Optional): At months 0 and 12,
10 mL of serum will be collected for storage for future analysis (non-
genetic) in participants who agreed to this. Serum may be collected up to
14 days after the visit if blood is unable to be collected at the time of the
visit.”
4. Adverse event grading (section 7.1) added bullet point three : “ All grade 3
and 4 adverse events judged to be associated with doxycycline (possibly,
probably or definitely related)”
2/24/2020 1. Clarified that specimens collected due to presumptive infection may be
version 5.0 sent to storage until the research team confirms an absence of STI, at
which point the sample will be discarded
2. Updated types of infection for resistance testing to revise definition
of syphilis from "primary syphilis with chancre" to "syphilis with
chance/mucosal lesion", and remove exposure to syphilis with
presumptive treatment
 72

3. Clarified that only swabs/urine samples obtained from anatomic site of

infection will undergo molecular resistance testing among those with
documented STIs

6/30/2020 1. Discussion of impact of COVID-19 pandemic on the study and plans to

version 6.0 address it
2. Added qualitative interview guide procedures for a subset of participants
3. Added procedures to re-enroll participants who had been enrolled in the
study prior to 3/15 and offer participation for an additional, continuous 12
months
4. Plans to monitor discontinuation rates among re-enrolled participants as
part of the statistical analysis
5. Updates to consenting procedures, including re-consenting participants
who had enrolled prior to 3/15 and use of electronic consenting
procedures such as DocuSign

11/20/2020 1. Changed the timeline for standard of care STI tests that can be used at
Version 7.0 enrollment, from 14 days to 30 days
2. Clarification of inclusion criteria related to STIs and syphilis diagnoses
3. Clarification on when testing for evidence of STIs can be conducted in
relation to STI treatment

3/20/2022 1. Added chlamydia culture for participants with pharyngeal or rectal

Version 8.0 chlamydia
2. Addition of DoxyPEP association of PrEP persistence and HIV virologic
suppression to exploratory objectives
3. Included month 12 swab for commensal Neisseria in all participants, not
just those with culture positivity at baseline
4. Added hematologic testing in standard of care arm to provide comparator
for doxyPEP arm

5/27/2022 1. Updated with DSMB findings from 5/2022 which resulted to early closure
Version 9.0 to enrollment
2. Closed to enrollment
3. Participants enrolled in the control arm offered study provided doxycycline
during remainder of study follow-up
4. Added qualitative interviews for providers
 73

STATISTICAL ANALYSIS PLAN

VERSION: 1.0
23 Feb 2021

BASED ON:
Protocol Version 5.0 February 24, 2020
Amendments and Dates
CRF Date:

STUDY TITLE:
DoxyPEP: Prospective, randomized, open-label, clinical trial to evaluate the impact of doxycycline
post-exposure prophylaxis on the occurrence of bacterial STIs among men who have sex with men
(MSM) and transgender women (TGW) PrEP users and people living with HIV
74

23/Feb/2021

23 Feb 2021
 75

PROTOCOL SUMMARY

Design:
Open label randomized clinical trial of doxycyline PEP to reduce bacterial STIs (N.
gonorrhea [GC], C. trachomatis [CT], and T. pallidum [syphilis]) among men who
have sex with men (MSM) and transgender women (TGW) living with HIV (PLWH)
or on HIV PrEP.

Study Population:
390 PLWH participants and 390 participants on HIV pre-exposure prophylaxis
(PrEP), for a total sample size of 780

Study Sites:
1. HIV/AIDS Clinic (“Ward 86”), Zuckerberg San Francisco General Hospital, UCSF,
San Francisco
2. San Francisco City Clinic, San Francisco Department of Public Health, San
Francisco (municipal STD clinic)
3. Madison HIV/AIDS Clinic, Harborview Medical Center University of Washington,
Seattle
4. Public Health-Seattle & King County STD Clinic at Harborview Medical Center,
Seattle

Primary Study Objectives:

1. Evaluate the effectiveness of doxycycline post-exposure prophylaxis (PEP) to
reduce STI infections in two populations: a) MSM/TGW living with HIV and b)
MSM/TGW taking HIV PrEP.
2. Investigate the impact of doxycycline PEP on development of culture-based
tetracycline (TCN) resistance in N. gonorrhea (GC) and among nasopharyngeal
carriers of S. aureus.

Secondary Study Objectives:

1. Assess the safety, tolerability, and acceptability of doxycycline PEP.
2. Investigate the impact of doxycycline PEP on development of molecular
markers of tetracycline (TCN) resistance in C. trachomatis (CT), and T. pallidum
(syphilis).
3. Evaluate the development of phenotypic tetracycline resistance among
nasopharyngeal carriers of commensal Neisseria species.
4. Measure the proportion of sex acts which are covered by doxycycline PEP.
5. Assess the effect of doxycycline PEP on the gut resistome through measuring
the abundance of tetracycline resistance genes in a subset of 50 MSM/TGW
living with HIV and 50 MSM/TGW on PrEP assigned to receive doxycycline PEP
as well as rectal swabs from all participants, stored for future evaluation.
 76

Exploratory objectives:
1. Evaluate the diversity of the gut microbiome and quantity and breadth of drug
resistance genes in participants on doxycycline PEP and not on PEP.
2. Assess the association between meningococcal vaccination and incident GC
infection.
3. Evaluate the incidence of nongonococcal, non-chlamydial urethritis and the
proportion of urethritis in which Mycoplasma genitalium (MG) is detected.
4. Assess the rate of detection of Mycoplasma genitalium (MG) in urine in each
arm and the presence of tetracycline and other antimicrobial resistance in MG
in each arm.
5. Evaluate the incidence of lymphogranuloma venereum (LGV) in those
diagnosed with rectal chlamydia in each arm.
6. Evaluate whether doxycycline PEP is associated with PrEP persistence among
persons on PrEP and HIV virologic suppression among persons on ART by
comparison of arms and within the doxycycline PEP arm by adherence to
doxycycline PEP.

Approach:
Eligible participants randomized to receive PEP will receive open-label doxycycline
200 mg to be taken ideally within 24 hours but no later than 72 hours after each
condomless sexual contact (receptive anal, insertive anal, vaginal, or oral, or use of
shared sex toys). Participants are instructed to take no more than 200 mg in each
24-hour period. Sexual activity and doxycycline PEP usage will be recorded using a
smartphone application and assessment at study visits. At enrollment and
quarterly for 12 months of follow-up, GC and CT testing will be conducted on
samples from the oropharynx, rectum and urine and a blood specimen for syphilis
serology will be collected. Resistance testing will be conducted on specimens that
test positive for GC using phenotypic methods, and molecular techniques for CT and
syphilis (for syphilis, T. pallidum DNA will attempt to be obtained from mucosal or
lesional swabs). Nares and pharyngeal swabs will be obtained to evaluate
tetracycline resistance among nasopharyngeal carriers of S. aureus and commensal
Neisseria species (oropharyngeal swab only). Stool samples from 50 MSM/TGW
living with HIV and 50 on PrEP in the doxycycline PEP arm at three time points will
undergo metagenomic sequencing for tetracycline resistance genes. Rectal swabs
from all participants who consent will be archived for future evaluation of the
enteric microbiome and resistome, as well as for presence of MG.
 77

TABLE OF CONTENTS
PROTOCOL SUMMARY .....................................................................................................................3
TABLE OF CONTENTS........................................................................................................................5
LIST OF ABBREVIATIONS ..................................................................................................................7
1. INTRODUCTION.............................................................................................................8
2. STUDY OBJECTIVES AND ENDPOINTS ...........................................................................9
2.1. Study Objectives ...........................................................................................................9
2.2. Study Endpoints ............................................................................................................9
3. STUDY DESIGN ........................................................................................................... 13
3.1. Summary of Study Design .......................................................................................... 13
3.2. Definition of Study Drugs........................................................................................... 13
3.3. Sample Size Considerations ....................................................................................... 14
3.4. Randomization ........................................................................................................... 15
3.5. Clinical Assessments .................................................................................................. 15
4. PLANNED ANALYSES .................................................................................................. 16
4.1. Interim Analyses ........................................................................................................ 16
4.2. Final Analyses............................................................................................................. 16
5. GENERAL CONSIDERATIONS FOR DATA ANALYSES AND HANDLING ........................ 17
5.1. Data Presentation Conventions ................................................................................. 17
5.2. Analysis Populations .................................................................................................. 17
5.3. Handling of Missing Data ........................................................................................... 18
6. STUDY POPULATION .................................................................................................. 19
6.1. Participant Disposition............................................................................................... 19
6.2. Screen Failures ........................................................................................................... 19
6.3. Protocol Deviations.................................................................................................... 19
6.4. Study Termination Status .......................................................................................... 19
6.5. Demographic and Baseline Characteristics ............................................................... 19
7. EFFICACY .................................................................................................................... 21
7.1. General Considerations ............................................................................................. 21
7.2. Statement of the Null and Alternate Hypotheses ..................................................... 21
7.3. Subgroup Analyses..................................................................................................... 21
 78

7.4. Multiple Comparisons and Multiplicity ..................................................................... 21

7.5. Analysis of the Primary Efficacy Endpoint ................................................................. 21
8. SAFETY AND TOLERABILITY ........................................................................................ 26
8.1. Adverse Events and Deaths ....................................................................................... 26
8.2. Laboratory Data ......................................................................................................... 27
8.3. Concomitant and Other Medications ........................................................................ 27
8.4. Tolerability ................................................................................................................. 27
8.5. Acceptability .............................................................................................................. 28
9. ADDITIONAL SECONDARY ENDPOINT ANALYSIS ....................................................... 29
9.1. Coverage of sex acts .................................................................................................. 29
9.2. Gut resistome sub-study............................................................................................ 29
10. REVISIONS TO THE SAP .............................................................................................. 30
11. APPENDIX ................................................................................................................... 31
11.1. Table of Contents for Data Reports ........................................................................... 31
12. REFERENCES ............................................................................................................... 33
 79

LIST OF ABBREVIATIONS
Table 1: List of Abbreviations
Abbreviation Term
AE Adverse Event
ART Antiretroviral therapy
CASI Computer Assisted Self-Interview
CRF Case Report Form
CT Chlamydia trachomatis
GC Neisseria gonorrhea
GCP Good Clinical Practices
ITT Intent-to-Treat Population
NAAT Nucleic acid amplification test
PEP Post-exposure prophylaxis
PrEP Pre-exposure prophylaxis
PP Per-Protocol Population
s.d. Standard Deviation
SAE Serious Adverse Event
TCN Tetracycline
 80

1. INTRODUCTION
The purpose of this document is to provide details on study populations and on how the
variables will be derived, how missing data will be handled as well as details on statistical
methods to be used to analyze the safety and efficacy data.
The document may evolve over time, for example, to reflect the requirements of protocol
amendments or regulatory requests. However, the final SAP will be finalized, approved by the
Protocol Chairs, and placed on file before the database is locked.
 81

2. STUDY OBJECTIVES AND ENDPOINTS

2.1. Study Objectives

2.1.1. Primary Objective
1. Evaluate the effectiveness of doxycycline post-exposure prophylaxis (PEP) to reduce STI
infections in two populations: a) MSM/TGW living with HIV and b) MSM/TGW taking HIV
PrEP.
2. Investigate the impact of doxycycline PEP on development of culture-based tetracycline (TCN)
resistance in N. gonorrheae (GC) and among nasopharyngeal carriers of S. aureus.

2.1.2. Secondary Objective

1. Assess the safety, tolerability, and acceptability of doxycycline PEP
2. Investigate the impact of doxycycline PEP on development molecular markers of
tetracycline (TCN) resistance C. trachomatis (CT), and T. pallidum (syphilis).
3. Evaluate the development of phenotypic tetracycline resistance among nasopharyngeal
carriers of commensal Neisseria species.
4. Measure the proportion of sex acts which are covered by doxycycline PEP
5. Assess the effect of doxycycline PEP on the gut resistome through measuring the
abundance of tetracycline resistance genes in a subset of 50 MSM living with HIV and 50
MSM/TGW on PrEP assigned to receive doxycycline PEP as well as rectal swabs from all
participants, stored for future evaluation.

2.2. Study Endpoints

2.2.1. Primary Endpoints

2.2.1.1. Effectiveness of doxycycline

The primary endpoint is any of the following STI endpoints post-enrollment:
• Documented positive GC by NAAT
• Documented positive CT by NAAT
• Early syphilis infection based on four-fold increase in non-treponemal titers or
positive darkfield on exudate from a lesion.
STIs are assessed at month 3,6, 9 and 12 visits in all participants, in addition to testing at interim
visits, if indicated (e.g. symptomatic). A primary endpoint event is defined as occurrence of
any one of the above STIs diagnosed within each quarter, with the exception that repeat
diagnosis of the same STI within 28 days of a quarterly visit is assumed to be the same infection
(i.e. not an incident infection). Any infection diagnosed at an interim visit (outside the 28 days)
will be counted as a primary endpoint for that quarter i.e., if a participant is diagnosed with GC
 82

at 40 days after their 6 month visit, this will be analysed as a primary 9 month endpoint. All
endpoints will be reviewed by the endpoint adjudication committee, blinded to arm
assignment, for final determination, using most current CDC STI guidance.

2.2.1.2. Impact of doxycycline on TCN resistance

1. Resistance of GC to TCN detected in positive GC cultures. Those with positive GC NAAT results
will have a swab obtained from the involved anatomic site and other sites of sexual contact in
past 90 days (to include throat, rectum, urine) for GC culture-based phenotypic testing. Swabs
for GC culture will be collected at time of suspected GC or in patients recalled after positive GC
NAAT result reported.
2. Tetracycline resistance detected in S. aureus culture-positive nasopharyngeal swabs, collected at
baseline, six, and 12 months

2.2.2. Secondary Endpoints

2.2.2.1. Efficacy of doxycycline for individual STIs

Effectiveness of doxycycline will also be assessed for each of the STIs separately, for both
cohort combined, using endpoints as defined above.

2.2.2.2. Safety, tolerability and acceptability of doxycycline PEP

• Safety: All grade 2 and higher hematologic and hepatic laboratory abnormalities, all
grade 3 and 4 adverse events judged to be associated with doxycycline (possibly,
probably, or definitely related), and all SAEs.
• Tolerability: in person quarterly assessments of discontinuations of doxycycline,
discontinuation of PEP, symptom assessment, and self-reported side effects
questionnaire at 6 and 12 months by CASI (computer assisted self-interview),
In person assessment by research staff at scheduled visits to include:
- All permanent doxycycline discontinuation and reasons for discontinuation;
- For all temporary doxycycline interruptions ≥7 days, reason for interruption; and
- Side effects:
o presence/absence of the following symptoms and if present, whether
attributed to doxycycline (if taking) by a) participant and b) investigator:
rash, sunburn, headache, change in vision, pain with swallowing,
diarrhea, nausea, vomiting, abdominal pain, or other.
CASI questions related to tolerability
 83

- At least once in the past 3 months, I did not take doxycycline within 3 days
after sex because I wanted to avoid side effects. (Reported using 5-point
Likert scale)
- Were there any other reasons that you did not take doxycycline within 3 days
after sex? Yes/No (Y/N) and open-ended response if yes is selected.
- In the past 3 months, do you feel that taking doxycycline affected your sexual
experience? (Reported using 3-point scale: negative, neutral, positive effect).
• Acceptability: self-reported acceptability questionnaire by CASI for all doxycycline
PEP recipients administered at month 6 and 12.
Answer to the following questions, reported using a 5-point Likert scale, unless
otherwise indicated
− Doxycycline is easy to take.
− I'm worried that taking doxycycline will cause antibiotic drug resistance. (reverse
coded)
− I'm worried that doxycycline will hurt my body. (reverse coded)
− Taking doxycycline PEP will reduce my condom use. (reverse coded)
− Doxycycline has other good effects for me.
− Taking doxycycline PEP has helped me take my PrEP or ART more regularly.
− A sexual partner has expressed negative feelings about me taking doxycycline.
(reverse coded)
− A sexual partner has expressed positive feelings about me taking doxycycline
− Taking doxycycline only when needed after sex and not every day is... (5-point
scale from very unacceptable to very acceptable)
− In my community, taking doxycycline to prevent STDs is considered... (5-point
scale from very unacceptable to very acceptable)
− I would continue to take doxycycline to reduce sexually transmitted infections if
available to me at no cost after the study.
− I would recommend doxycycline PEP to a friend. (Y/N)

2.2.2.3. Sex acts covered by doxycycline PEP

Sex acts covered by doxycycline will be recorded by quarterly CASI assessment and medication
reconciliation as well as optional usage of an ongoing smartphone app (Blackbook).
• CASI: Coverage of sexual contacts and accompanying doxycycline use assessed at
month 6 and 12 in the doxycycline PEP arm assessed by the following questions:
− In the past 3 months, I missed taking doxycycline within 3 days after sex at least
once. Y/N
− In the past 3 months, when you have had anal or vaginal/frontal without using a
condom the whole time, how often did you take doxycycline PEP within 72 hours
after sex? 5-point scale
 84

− In the past 3 months, when you have given or received oral sex, how often did
you take doxycycline PEP within 72 hours after oral sex? 5-point scale
-- Of the TIMES you had anal sex or vaginal/frontal sex without a condom, how
many TIMES did you take doxycycline PEP within 72 hours after sex? Numeric
value
− The last time you had anal or vaginal/frontal sex without using a condom the
entire time, did you take doxycycline? Y/N
− Of these times you had oral sex, how many TIMES did you take doxycycline PEP
within 72hrs? Numeric value
− The last time you had oral sex did you take doxycycline. Y/N
− How many times did you take doxycycline PEP more than 72 hours after anal or
vaginal sex?
− How many times did you take doxycycline PEP more than 72 hours after oral
sex?

• Quarterly review of doxycycline dispensing and remaining tablets:

− Number of doxycycline pills returned at each quarterly visit.
− Bottles of doxycycline dispensed at each visit.
• Smartphone app: Coverage of sex acts by doxy will also be assessed by use of app,
which will prompt participants to record all sexual activity (oral, anal, vaginal), use of
condoms, and whether doxycycline was taken after qualifying sexual contact
(proportion of sex acts covered).

2.2.2.4. Tetracycline resistance

• Molecular markers of resistance for CT
• Molecular markers of resistance to syphilis
• Phenotypic resistance in nasopharyngeal swabs culture positive for Neisseria
• Abundance of TCN resistant genes in rectal swabs from MSM/TGW receiving
doxycycline PEP (based on total Tet C and Tet M genes detected, normalized for the
quantity of bacterial present, not isolated from specific flora)
 85

3. STUDY DESIGN

3.1. Summary of Study Design

Open label randomized clinical trial of doxycyline PEP to reduce bacterial STIs (N. gonorrheae
[GC], C. trachomatis [CT], and T. pallidum [syphilis]) among MSM/TGW) living with HIV (PLWH)
or on HIV PrEP. The trial is powered to separately assess impact for MSM/TGW living with HIV
and MSM/TGW on PrEP.

Eligible participants randomized 2:1 to doxycycline PEP will receive open-label doxycycline 200
mg to be taken ideally within 24 hours but no later than 72 hours after condomless sexual
contact ((receptive anal, insertive anal, vaginal, or oral, or use of shared sex toys).). 200 mg of
doxycycline will be taken at most once per 24-hour period regardless of the number of sexual
acts occurring during this time period. Sexual activity will be recorded for both arms of the
study (doxycycline PEP and control condition) by the participant using a smartphone application
that will be adapted for study use; this will enable comparable assessment of risk in the two
arms. PEP pill-taking will also be measured by the app to enable assessment of coverage of sex
acts by PEP. Sexual activity and adherence will also be assessed in person at quarterly visits.

STI testing will be conducted quarterly from three anatomic sites (pharyngeal, rectal, and urine
samples for GC and CT NAAT testing) and blood obtained for syphilis testing, following CDC
guidelines for serologic diagnosis of syphilis.

Participants with a positive STI test will return for STI treatment and for swabs of the affected
site for resistance testing; culture- and molecular-based for GC and molecular methods for GC,
CT and syphilis. Those with signs and symptoms suggestive of syphilis infection and those with a
reactive syphilis serologic test that indicates a new syphilis infection will have swabs of any
current active lesion (if present) as well as mucosal swabs from the oropharynx to obtain DNA
for molecular testing.

To assess tetracycline resistance in a common, sometimes pathogenic bacteria, swabs from the
anterior nares and oropharynx will be obtained at baseline, 6 and 12 months to identify S.
aureus carriers in whom tetracycline resistance will be evaluated. To assess tetracycline
resistance that could be transferred to oropharyngeal GC from commensal Neisseria species,
oropharyngeal swabs will be obtained at baseline and 12 months to evaluate tetracycline
resistance in a carrier.

3.2. Definition of Study Drugs

The study drug is open-label doxycycline 200 mg to be taken ideally within 24 hours but no later
than 72 hours after condomless sexual contact. Participants will be instructed to take 200 mg
of doxycycline at most once per 24-hour period regardless of the number of sexual acts
occurring during this time period.
 86

3.3. Sample Size Considerations

3.3.1. Sample Size Justifications
The sample size of 390 MSM/TGW in each cohort (MSM/TGW living with HIV and MSM/TGW
on PrEP) has 80% power with 0.05 two-sided Type 1 error, for the endpoint of any lab-detected
incident early syphilis, GC and/or CT infection, based on quarterly assessments for each person,
10% annual loss to follow-up and an intra-class correlation of 0.2. With these assumptions, a
cohort of 390 of MSM/TGW living with HIV and 390 of MSM/TGW on PrEP (130 in the control
and 260 in the intervention for each cohort) will provide 80% power to detect a decrease in
quarterly STI prevalence from 10% to 5%, which corresponds to an annual reduction in
combined STI incidence from 34% to 19%, assuming effective treatment.
We expect in the control arm approximately 5% of participants to have GC, 5% CT and 2% early
syphilis at each quarterly visit; some will be diagnosed with more than one infection. We
anticipate effectiveness of doxycycline PEP to be 35% for GC, 65% for CT and 65% for syphilis.
Sample sizes were computed using nQuery for repeated measures for two proportions,
assuming four visits for each participant, and ratio estimators for the specified reduction.
Approximately 10% annual loss to follow-up is assumed, because of partial information
expected at some visits, this is approximated with sample size of 370.
Table 2 Power Table for N = 390; 2:1 randomization doxycycline PEP: SOC

Power to detect 50% Power to detect 65%

Control arm Intervention arm
reduction reduction
quarterly quarterly
prevalence prevalence
ICC = 0.2 ICC = 0.1 ICC = 0.2 ICC = 0.1

10% 5% 80% 87% 96% 99%

7.5% 3.75% 68% 76% 90% 95%

5% 2.5% 51% 59% 77% 85%

3% 1.5% 35% 41% 57% 65%

2% 1% 26% 30% 43% 50%

3.3.2. Sample Size Re-estimation

At the point when enrollment is near completion, the study team will evaluate the expected
power of the trial, based on the observed rate of infections, estimating intra-class correlation
and retention to date in the trial. If the rate of the primary endpoint is substantially lower than
anticipated, sample size re-estimation would be used to restore power to the extent possible,
through additional enrollments, increase in person-time, or modification of risk criteria.
 87

3.4. Randomization
The randomization is 2:1 for assignment to doxycycline PEP versus standard of care (no
doxycycline PEP). Randomization is stratified by four sites, two primarily enrolling HIV-infected,
and two HIV-uninfected participants, with block sizes selected with size randomly selected from
between 15 to 30.

3.5. Clinical Assessments

Safety assessments are limited to the following:
• All grade 2 and higher hematologic and hepatic laboratory abnormalities that are
attributed to doxycycline in the opinion of the site investigator
• All AEs meeting SAE definition
• All grade 3 and 4 adverse events judged to be associated with doxycycline (possibly,
probably or definitely related)
In this open label trial, for those randomized to receive doxycycline PEP, all AEs and SAEs have
attribution recorded as doxycycline-related or not doxycycline-related, in the judgment of the
site investigator.
 88

4. PLANNED ANALYSES

4.1. Interim Analyses

The trial will be monitored by an independent data monitoring committee approximately every
6 months. A single formal interim monitoring time is planned, when approximately half of the
planned total enrollment have completed the 6-month visit, with stopping rules based on the
achieved accrual of primary outcomes using the O’Brien-Fleming boundaries. Each cohort will
be monitored separately. Stopping for both efficacy and lack of efficacy will be considered.
However, a decision to stop for efficacy will need to balance the need for evidence assessing
the potential harm of doxycycline-associated side effects.

4.2. Final Analyses

The final analysis will be conducted after database lock of all final (12 month) study visits.
 89

5. GENERAL CONSIDERATIONS FOR DATA ANALYSES AND HANDLING

5.1. Data Presentation Conventions

Continuous variables (e.g., age) are summarized using descriptive statistics (the number of
subjects with available data, the mean, standard deviation, median and minimum and
maximum). Categorical variables (e.g., race) are summarized using counts and percentages.
Percentages are calculated using the total subjects with data per treatment group.

5.2. Analysis Populations

5.2.1. ITT population
The intent-to-treat (ITT) population comprises all randomized participants. There are two ITT
populations: HIV-uninfected MSM and TGW on PrEP; HIV-infected MSM and TGW.

5.2.2. Safety population

The ‘Safety Population’ is defined as all subjects who receive at least one dose of study
medication. This population is expected to be identical to participants randomized to
doxycycline PEP for this study.

5.2.3. Blackbook responders

The subset of the ITT population that participates (i.e. contributes data) in the Blackbook app.

5.2.4. GC positive population

Post-baseline GC positive participants assessed for TCN resistance. Participants with positive
GC will be assessed for culture based TCN resistance and for molecular resistance.

5.2.5. CT positive population

Post-baseline CT positive participants assessed for TCN resistance. Participants with positive CT
will be assessed for molecular-based TCN resistance.

5.2.6. Syphilis positive population

Post-baseline Syphilis positive participants assessed for TCN resistance. Participants with
incident syphilis will be assessed for molecular-based TCN resistance.

5.2.7. Nasal or oropharyngeal S. aureus carrier Population

Participants with nasopharyngeal cultures with S. Aureus growth assessed for TCN resistance.

5.2.8. Neisseria positive population

Participants with nasopharyngeal Neisseria growth assessed for oropharyngeal TCN resistance.
 90

5.2.9. Hair population

Participants who have hair assessed for presence of doxycycline.

5.2.10. Gut resistome Population

50 HIV positive MSM and 50 HIV negative MSM/TGW assigned to the doxycycline PEP arm
assessed for tetracycline resistance using rectal swabs.

5.2.11. Subgroups
The following are subgroups of interest:
• MSM only
• Age (< 30 vs. > 30)
• City (San Francisco/Seattle)
• Use of HIV PrEP (Event driven/Daily)

5.3. Handling of Missing Data

5.3.1. Missing Efficacy Endpoints
Complete case analysis will be used for the primary efficacy analysis, unless substantial
imbalance (> 8%) in missing primary endpoint by arm occurs. Appropriate missing data
methods (e.g. imputation) or causal estimation of direct effects will be conducted to
assess the potential influence of differential missingness by arm.
 91

6. STUDY POPULATION

6.1. Participant Disposition

A consort diagram will describe for each cohort separately the number screened, randomized,
retained by visit, and contributing to the primary analysis endpoints by arm. Reason for screen-
out and non-inclusion in the primary outcome will be summarized.

6.2. Screen Failures

Reason for screen failures will be summarized by arm and by site.

6.3. Protocol Deviations

A listing or summary of major protocol Deviations will be provided.

6.4. Study Termination Status

Reason for early study termination or study withdrawal will be enumerated and detailed. Study
disposition at the time of a report will list those remaining in study follow-up, completed study
follow-up, and early withdrawal (including deaths).

6.5. Demographic and Baseline Characteristics

The following baseline demographics and risk characteristics will be presented separately by
cohort, by arm, and by site for the ITT cohort:
• Age in years
• Race/ethnicity
• Education
• Gender identity
• Sexual preference
• Sexual risk
− Number and HIV serostatus of partners
− Frequency of oropharyngeal, penile, and rectal exposure with and without
condoms
− STI(s) in year before enrollment
• alcohol use
• meth use
• IDU
• recreational drug use
• Living situation
• Health insurance
• Currently taking PrEP (Daily vs. event driven)/ART
 92

6.5.1. Baseline Laboratory Data

Baseline bacterial STI diagnoses (GC/CT, Syphilis) detected by lab-based study testing will be
reported for each cohort separately, by arm and by study site. Infections will be tabulated by
sites(s) of infections
 93

7. EFFICACY

7.1. General Considerations

Tabulations will be by study arm within each cohort, for each quarter of study follow-up (or at
visits where evaluated). These will be reported in only the closed report during the study.
Parallel tabulations will be given by study site only if substantive differences are observed
between sites (which are not expected).
Inferential statistical tests will be two-sided and will be performed at alpha levels of 0.05 and
0.10 to declare the significance of main effects and interaction effects, respectively.
Analyses will be conducted separately for HIV-infected and HIV-uninfected cohorts for the
primary endpoint, and for the combined cohort. All secondary endpoints will be conducted in
the combined cohort.

7.2. Statement of the Null and Alternate Hypotheses

For both HIV-uninfected MSM/TGW on PrEP and HIV-infected MSM/TGW separately:
• Ho: Use of doxycycline PEP does not have a differential effect on incidence of GC, CT
and syphilis infections compared to the standard of care.
RR any STI (Doxy Arm/SOC Arm) = 1
• HA: Use of doxycycline PEP does have differential effect the incidence of GC, CT and
syphilis infections compared to the standard of care.
RR any STI (Doxy Arm/SOC Arm) ≠ 1

7.3. Subgroup Analyses

Subgroup analysis of the efficacy of the intervention will be conducted for
• Age subgroups (</> 30).
• Report of STI diagnosis in prior year.
• Baseline viral suppression (< 50 copies) for HIV-infected men.
• On PrEP at baseline (Daily/Event driven/No) for HIV-uninfected men.

7.4. Multiple Comparisons and Multiplicity

No adjustments are planned for multiple comparisons.

7.5. Analysis of the Primary Efficacy Endpoint

7.5.1. Primary Efficacy Analysis
The primary analysis of efficacy will be conducted in the ITT cohort.
Endpoint definitions:
 94

Primary endpoint: Any STI in the first, second, third or fourth quarter of follow-up, detected in
post-baseline testing up to and including STI testing at months 3,6,9 and 12. Any STI diagnosed
within 28 days of the same STI present at a quarterly visit will not be defined as an incident
infection (suggesting incomplete treatment or persistent NAAT positivity without active
infection, rather than re-infection). Incident infections detected and treated at interim visits
after 28 days will be defined as incident infections for the current quarter. To avoid potential
assessment bias, these will be handled in the analysis as if they would have been detected at a
scheduled visit (months 3, 6, 9, and 12). Therefore, an STI diagnosed at 40 days after the month
6 visit will be carried forward as a primary event at the next scheduled visit (month 9). If a
participant has more than one positive STI (either of the same or different bacterial infections
at a scheduled visit it will be counted as one primary event for that quarter. If scheduled visits
are missed, infections will be assigned to the next scheduled visit.
All endpoints will be reviewed by the endpoint adjudication committee, blinded to doxycycline
PEP assignment, for final determination, using most current CDC STI guidelines.

The criteria for an incident syphilis case will include a four-fold increase in non-treponemal
titers (e.g., the RPR) and/or a consistent clinical presentation (e.g., characteristic chancre in
primary syphilis which would be confirmed by a darkfield or reactive RPR without the
requirement for a four-fold increase in titer).

Descriptive analysis
• The number of participants with any bacterial STI, and with each type of STI, will be
summarized at each scheduled visit by arm.
• The number of STIs reported by each participant will be summarized at each
scheduled visit by arm.
• For CT and GC, the anatomical site of infections will be detailed.
Statistical analysis
Comparison of STI incidence by arm will be conducted by estimating the relative risks of any STI
over the 3, 6, 9, and 12 month visits using a modified Poisson model fitted using GEE methods
to account for repeated observations from each participant, assuming an independent
covariance structure, with site and study arm as the only covariates[1]. The test for significance
will be a two-sided alpha = 0.05. 95% confidence intervals will be computed using robust
standard errors.
The same analysis will be repeated for the individual STIs, potentially also by anatomical site.

7.5.2. Sensitivity Analyses of the Primary Efficacy Results

Several sensitivity analyses are planned:
1. Time to first STI using Kaplan-Meier and Cox PH regression.
 95

2. Including all STIs diagnosed, irrespective of timing. The primary outcome will be
modified to include any STI re-diagnosed within 28 days of the scheduled quarterly
visits, and the statistical analysis for the primary analysis repeated.
3. Analysis counting each separate STI diagnosis as an event. A Poisson model fitted using
GEE methods to account for multiple events from each participant will be used to
estimate the change in rate of infections between arms. If necessary, to achieve a
better fit, a zero-inflated Poisson model may be used.

7.5.3. Analyses of the Primary Efficacy Results while on study drug (Per protocol)
For the per-protocol analysis, for each cohort the doxycycline PEP arm will be restricted to
study time prior to the first permanent discontinuation of study drug by self-report or
documented on the study CRFs.
First discontinuation is defined as first of
• Product discontinuation
• Missed quarterly visit
Descriptive: Occurrence and reasons for first discontinuation will be tabulated (number of
participants).
Analysis: The evaluation of the impact of non-disrupted use of doxycycline on any STI will use
the same model as specified for the primary analysis, but also potentially adjusted for age,
race/ethnicity, baseline STI, and baseline and follow-up sexual risk (number of partners,
unprotected anal sex, employment). For the HIV negative cohort, PrEP at baseline; for the HIV
positive cohort, viral suppression at baseline will also be considered.

7.5.3.1. Study drug use and time on study drug in doxycycline PEP arm
The following descriptive tabulations will be completed for each measure:
• Study drug dispensation (number of participants and number of pills) will be
summarized for each study visit over time by cohort.
• Proportion of sex acts and condomless sex acts where doxycycline was taken, based
on self-report will be tabulated for each quarter and overall within each cohort.
Evaluated for both the mobile app (Blackbook cohort only) and CRF calendar data.
• Semiquantitative assessment (high/low) of concentrations in hair will be tabulated
for each quarter and overall within each cohort (Hair cohort only).
• Concomitant use of doxycycline, as documented in the antibiotic use log, will be
summarized for the control and doxycycline arms
Descriptive comparisons are planned to evaluate the consistency of measures across different
measurement modalities
• Mobile app, hair, and CASI data will be compared using variables defined to
approximate the same time period.
 96

7.5.3.2. Assessment of doxycycline in hair

Longitudinal exposure to doxycycline assessed using hair collection.
• A semiquantitative evaluation in the control arm (doxycycline present vs. absent).
• A semiquantitative assessment in the PEP arm (doxycycline concentration high vs.
low vs. none).
Descriptive: The proportion of samples with doxycycline present, and the concentration levels
will be tabulated for each quarter and overall by study arm within each cohort.

7.5.4. Secondary analysis of efficacy for each site of infection

Secondary endpoints:
• Any GC.
• Any CT.
• Any new early syphilis diagnosis.
• Site of GC infection (pharyngeal GC compared to urine or rectal GC).
• Site of CT infection (pharyngeal CT compared to urine or rectal CT).
Analysis: For secondary endpoints, the same approach as defined for the primary endpoint will
be used, restricted to the specific bacterial infections or infection site.

7.5.5. Analysis of Tetracycline resistance

7.5.5.1. Primary analysis of TCN resistance in GC cases

The primary analysis of TCN resistance in GC will be conducted in the GC positive.
Descriptive statistics: Tabulations of resistance amongst cases (GC: TCN resistance testing
missing, not resistant, resistant) will be reported at baseline, month 3, 6, 9, and 12 (and
potentially interim visits) in each arm, for any infection site, and broken out by infection site.
Additional descriptive information may also be included (i.e. change in MIC in GC cultures
amongst those with repeat infections)
Statistical analysis: Assessment of odds of resistance amongst GC cases with resistance results
will be assessed by arm using logistic regression with arm as exposure, adjusted for city. To
account for repeated infections, repeated measures methods (GEE) will be used. Analysis
comparing arms also potentially adjusted for age, race/ethnicity, baseline STI, baseline and
follow-up sexual risk (number of partners, unprotected anal sex, employment). For the HIV-
negative cohort, PrEP at baseline; for the HIV-positive cohort, viral suppression at baseline will
also be considered.
 97

7.5.5.2. Analysis of TCN resistance in CT cases

The analysis of TCN resistance in CT cases will be conducted in all persons with a positive CT
diagnosis after baseline.
Outcome: Molecular TCN resistance will be defined as presence of mutations associated with
TCN resistance in the CARD database.
Descriptive statistics: Tabulations of TCN resistance in CT cases (TCN resistance testing missing,
not resistant, resistant) will be reported at baseline, 3-, 6-, 9-, and 12-month visits in each arm.
Statistical analysis: Assessment of difference in prevalence of TCN resistance by arm across all
post-baseline visits combined will be assessed by estimating the relative risk of TCN resistance
using logistic regression. The model will assess the relative odds of resistance by arm, adjusted
for study site.

7.5.5.3. Analysis of TCN resistance in Syphilis cases

The analysis of TCN resistance in Syphilis cases will be conducted in all persons with an incident
syphilis cases after baseline.
Outcome: Molecular TCN resistance will be defined as presence of mutations associated with
TCN resistance in the CARD database.
Descriptive statistics: Tabulations of TCN resistance in Syphilis cases (TCN resistance testing
missing, not resistant, resistant) will be reported at baseline, 3-, 6-, 9-, and 12-month visits in
each arm.
Statistical analysis: Assessment of difference in prevalence of TCN resistance by arm across all
post-baseline visits combined will be assessed by estimating the relative risk of TCN resistance
using modified Poisson regression. The model will assess the relative risk of resistance by arm,
adjusted for study site.

7.5.5.4. Analysis of TCN resistance in S. aureus carriers

The primary analysis of TCN resistance will be conducted in S. aureus cases detected at
Baseline, 6- and 12-month visits.
Outcome: TCN resistance will be a dichotomous outcome defined as sensitive or resistance,
based on e-Test cut-offs.
Descriptive statistics: Tabulations of TCN resistance in S. aureus cases (TCN resistance testing
missing, not resistant, resistant) will be reported at baseline, 6- and 12-month visits in each
arm. (site of S. aureus, if found, will not be known).
Statistical analysis: Assessment of difference in prevalence of TCN resistance by arm at each of
6 and 12 months will be assessed by estimating the relative risk of TCN resistance using
modified Poisson regression. The model will assess the relative risk of resistance by arm,
adjusted for study site at each time point. If an effect is found, assessment of a trend in time
will be assessed by fitting a linear trend over study time. If prevalence of resistance is < 5%,
analysis will pool across visits.
 98

8. SAFETY AND TOLERABILITY

Statistical tests are not planned for adverse event comparisons between arms. Safety and
tolerability will be assessed in the ITT cohort, or the doxycycline PEP arm only, given the open
label nature of the cohorts.

8.1. Adverse Events and Deaths

8.1.1. AE Definitions
Only the following AEs are recorded on eCRFs:
• All grade 2 and higher hematologic and hepatic laboratory abnormalities.
• All SAEs.
• In those randomized to doxycycline PEP arm, all grade 3 and 4 AEs with attribution
to doxycycline in the opinion of the site investigator.

8.1.2. Adverse Event Summary Tables

The number of Grade 2 and higher hematologic and hepatic AEs data will be tabulated by grade
and arm. Subjects will be counted only once for each hematologic and hepatic report. For the
doxycycline PEP arm, relatedness will be tabulated (see below of definition of relatedness).

8.1.3. Listings of Serious Adverse Events (SAE), Adverse Event Dropouts, and Death
SAE data will be reported as related and not related. The two categories will be defined as
follows:
• Related combines the options definitely, probably, and possibly
• Not related is not related.
Participant data listing for SAEs will be by cohort, and arm, sorted by reported date within
participant. Unique information included on the first page of the listing contains SAE onset and
resolution dates and times; verbatim description of SAE; severity and relationship to study
medication, action taken, and outcome.
A tabulation will present participants who discontinue study drug prematurely for the
doxycycline PEP arm due to an adverse event, and the reasons for discontinuations:
• Requirement for prohibited concomitant medications or other contraindication to
doxycycline.
• Occurrence of an adverse event requiring discontinuation of doxycycline.
• Request by participant to terminate study treatment.
• Clinical reasons believed life threatening by the physician, even if not addressed in
the toxicity section of the protocol.
• Requirement for chronic tetracycline use (>14 days).
 99

8.2. Laboratory Data

Laboratory data collected in this study consist of hematologic and liver function tests at months
3 and 9 in those on doxycycline PEP arm:
• Complete blood count (CBC) (white count, hemoglobin, platelets).
• Liver function testing (AST, ALT, total bilirubin and alkaline phosphatase)
The presentation of laboratory results focuses on the change from baseline to 3 and 9 months.
All participants in the doxycycline PEP arm who have a baseline and at least one follow-up
laboratory assessment, unless permanently discontinued doxycycline PEP, will be included in
the presentation of the laboratory data. For each laboratory test, descriptive statistics will
summarize the results at baseline, 3-, and 9-month assessment, and the change from baseline
to 3- and 9-months assessments. The summary statistics are N, mean, s.d., median, and range.
The within-group p-value from the paired t-test on the mean change from baseline and the 95%
confidence interval limits for the mean change from baseline are also included with the change
descriptive statistics. Figures will also be used to display change from baseline to 3 and 9
months.
The quantitative laboratory data for participants who have a baseline and follow-up laboratory
assessment may be presented as scattergrams plotting the change from baseline as a function
of the baseline value.
This analysis will omit values occurring after a participant has permanently discontinued study
drug.

8.3. Concomitant and Other Medications

8.3.1.1. Tabulation of time on PrEP and ART
Tabulations of PrEP and ART will use the following assessments:
• Adherence to HIV medications as reflected in viral suppression among participants
living with HIV, assessed by chart review of HIV RNA levels conducted as standard
clinical care and reported on CRFs.

8.4. Tolerability
Tolerability is assessed through:
• Self-reported response to whether a participant did not take doxycycline because of
side effects.
• Report of early termination due to side effects of study drug.
Descriptive: The Likert scale responses will be tabulated for each study visit for each cohort.
 100

8.5. Acceptability
Acceptability is assessed in the doxycycline PEP arm only via a seven-question acceptability
questionnaire using a 5-point Likert scale.
Descriptive: The scales will be converted to an acceptability score—using reverse coding where
appropriate—and summarized by cohort, site, and visit. Individual questions will also be
reported in detail.
 101

9. ADDITIONAL SECONDARY ENDPOINT ANALYSIS

9.1. Coverage of sex acts

Coverage of sexual contacts and accompanying doxycycline use is assessed by CASI at 6 and 12
months, and also assessed in the mobile app.
These self-report endpoints reflect coverage:
1) In the past 3 months, I missed taking doxycycline within 3 days after sex at least
once. Y/N
2) In the past 3 months, when you have had anal or vaginal/frontal without using a
condom the whole time, how often did you take doxycycline PEP within 72 hours
after sex? 5-pointscale
3) In the past 3 months, when you have given or received oral sex, how often did you
take doxycycline PEP within 72 hours after oral sex? 5-point scale
4) The last time you had anal or vaginal/frontal sex without using a condom the entire
time, did you take doxycycline? Y/N
5) The last time you had oral sex did you take doxycycline. Y/N
Descriptive: Each endpoint will be tabulated by visit for the doxycycline PEP arm. Five-point
scales will be dichotomized by combining “always/most of the time” vs. categories that are less
often.
Blackbook: App-acquired data will be used to compute the proportion of sex acts covered by
doxycycline PEP for each participant for all periods when the app was used, and the participant
remained on study drug.
Descriptive:
1) Participant use of the app will be summarized: number/proportion using the app,
number of weeks in use, compared to number of weeks of doxycycline PEP, proportion
of time described by App use; also summarized by study quarter.
2) Proportion of sex acts covered by doxycycline PEP for all person-time collected in each
quarter will be summarized.

9.2. Gut resistome sub-study

Analysis of the gut resistome will be described in a separate statistical analysis plan.
 102

10. REVISIONS TO THE SAP

Not Applicable
 103

11. APPENDIX

11.1. Table of Contents for Data Reports

Appendix to be completed
Title Population Comments

Study Population Section

ITT Unique
ITT Unique
ITT Unique
ITT Repeat 9.3.1
Efficacy Section
Figures
ITT Unique
ITT Unique
Tables
ITT Unique
ITT Unique
ITT Repeat
12.1.2.1
ITT Unique
ITT Unique
ITT Unique
Safety Section
Safety Unique
Safety Unique
Safety Unique
Safety Unique
Safety Unique
 104

Listings
Title Population Comment
Subject Enrollment Information Enrolled Unique
Subject Disposition Safety Unique
Subjects who did not Satisfy Inclusion/Exclusion Criteria Screening Failure Unique
(if applicable)
Protocol Deviations Safety Unique
Listing of Relationship of Serious Adverse Event Body Safety Unique
Systems, Group Terms, and Verbatim Text
 105

12. REFERENCES
1. Yelland LN, Salter AB, Ryan P. Performance of the modified Poisson regression approach
for estimating relative risks from clustered prospective data. Am J Epidemiol. 2011;174(8):984-
92. Epub 2011/08/16. doi: 10.1093/aje/kwr183. PubMed PMID: 21841157.