Clinical trial NCT05027516
Conditions: Neisseria Gonorrhoeae
Sponsor: Institute of Tropical Medicine, Belgium
Start date: 2022-01-17; Completion date: 2022-05-09
Enrollment: 42 participants; Allocation: RANDOMIZED; Intervention model: PARALLEL

Brief summary:
In this Randomized Controlled Trial, the investigators will recruit 42 men attending the STI and HIV clinic at the Institute of Tropical Medicine, with a diagnosis of N. gonorrhoeae and randomize them 1:1 to receive either ceftriaxone or ceftriaxone/azithromycin. They will be followed-up for a test of cure visit at day 14 post-treatment where urine, oropharyngeal and anorectal samples will be taken to test for cure and monitor treatment effects on the microbiome and resistome. The primary outcome will be evaluating the difference in the abundance of resistance conferring genes in the rectal microbiome in the two arms, 14 days after the receipt of therapy.

Detailed description:

Arms: arm 1: ceftriaxone 1g + lidocaine 35mg; intramuscular injection arm 2: ceftriaxone 1g + lidocaine 35mg intramuscular injection + azithromycin 2g orally
Interventions: intervention 1: Rocephin intervention 2: Azithromycin
Intervention descriptions: intervention 1: Single dose of Rocephine® (= ceftriaxone 1g + lidocaine 35mg) intramuscular injection intervention 2: Single dose of Rocephine® (= ceftriaxone 1g + lidocaine 35mg) intramuscular injection + single dose of azithromycin 2g orally

Protocol documents:
ICF_002.pdf: 
 
 
ResistAZM – ICF Eng. Version 1.1, dated 3Sept2021 
 
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Part 1. RESEARCH PARTICIPANT INFORMATION SHEET 
 
ResistAZM study:  
An open label randomized controlled trial comparing the effect of ceftriaxone 
plus azithromycin versus ceftriaxone for the treatment of Neisseria 
gonorrhoeae on the resistome  
 
Investigator:  
Dr. Chris Kenyon 
Sponsor: 
 
Institute of Tropical Medicine (ITM), Antwerp, Belgium 
Clinical site:  
ITM HIV/STI clinic 
Funder: 
 
Institute of Tropical Medicine (ITM), Antwerp, Belgium 
 
You are invited to take part in a research study which will be conducted in the HIV/STI clinic of the 
Institute of Tropical Medicine (ITM) and which seeks to contribute to preventing the emergence of 
resistance in gonorrhea and other bacteria. 
Before you decide whether to participate or not in this study, it is important that you understand the 
information in this form, because it explains your rights, our responsibilities to you, the purpose, 
procedures, possible benefits, risks and inconveniences related to this study, and the right to refuse 
or stop your participation at any time.  
Please, feel free to ask any questions at any time, for example about the possible benefits and possible 
risks related to this study. Your participation is completely voluntary. You can talk to anyone you trust 
about the research and you can take your time to think about whether you want to participate or not. 
In case you decide to participate, make sure you keep this background information and the signed 
consent form with you throughout the entire study period. 
 
PURPOSE AND DESCRIPTION OF THE STUDY 
Gonorrhea is a sexually transmitted infection (STI) which is caused by a bacteria (N. gonorrhoea) and 
which can manifest itself in the mouth/throat (pharyngeal gonorrhea), the rectum, or the genitals. 
This can occur with or without symptoms. Normally, any form of gonorrhea is treated with a single 
administration of antibiotics (ceftriaxone and azithromycin) and a check is performed after 14 days to 
see if the infection has disappeared. 
 
There is a however a real possibility that gonorrhea may become untreatable with antibiotics in the 
near future. This is caused by resistance of the gonorrhea bacteria, which is a change over time of the 
bacterial characteristics which makes that the bacteria no longer respond to the antibiotics treatment. 
As a result, the infection is harder to treat and there is an increased risk of spreading of the diseases 
and severe illness.  
This research study is done to better understand how gonorrhea is able to so rapidly develop 
resistance to antibiotics. In particular we want to see if gonorrhea is able to take up resistance 
mechanisms from other naturally occurring bacteria in your gut and in your mouth. When you get 
antibiotics this increases the amount of resistance genes in these bacteria that live in your gut, mouth 


 
 
ResistAZM – ICF Eng. Version 1.1, dated 3Sept2021 
 
p. 2 of 8 
and elsewhere. We want to test if gonorrhea is able to use these resistance genes from the bacteria 
that naturally occur in your body to become resistant to antibiotics.  
 
In this study, around 42 participants will take part in the ITM HIV/STI clinic in Belgium. Half of the 
participants will receive a treatment with a single antibiotic ‘ceftriaxone’ and the other half of the 
participants will be treated with a combination of 2 antibiotics: ‘ceftriaxone’ and ‘azithromycin’. 
Participants will be randomly allocated into one of the 2 treatment groups. Both these treatments are 
approved by the Belgian regulatory authorities for the treatment of a gonorrhea infection and are 
widely used in routine practice. The main research question we will be testing is that treatment with 
2 antibiotics results in more resistance than treatment with one antibiotic. If we find that 2 antibiotic 
therapy results in more resistance, then we will use this evidence to promote treatment with one 
antibiotic. Independent whether you will be treated with 1 or 2 antibiotics, you will receive a good 
treatment which is expected to cure your infection. Both the treatments with one or two antibiotics 
are close to 100% effective at eradicating gonorrhoea and both are recommended by national and 
international treatment guidelines. 
 
HOW THE STUDY IS DONE 
If you accept to be part of this study and if you meet the conditions to participate, you will be asked 
to attend 2 study specific visits (the current visit and a 2nd visit in 14 days), where the following 
procedures will be performed: 
 
Visit 1 = Today Visit 
The study doctor will ask you some standard medical questions (such as your antibiotic use in last 
year) as well as details on your lifestyle, including your sexual behavior. 
As part of your standard treatment, we will take some samples from you today, in order to fully assess 
your gonorrhea infection. Some additional samples will also be taken for the purpose of this study: 
• 
A urine sample 
• 
An anal swab 
• 
An oral rinse sample 
You will then have the usual treatment for gonorrhea which is an injection of ceftriaxone 1g alone or 
in combination with 2g of oral azithromycin depending on the group you will randomly be assigned.  
 
Visit 2 = Visit on day 14 (range day 13-15) 
You will be asked to come back to this clinic after two weeks. This is a routine visit for all cases of 
gonorrhea and this visit is to check whether you have been cured. You will be asked some questions 
about how you have been, whether you have had any other health problems since having the 
treatment, about your sexual behavior, lifestyle and antibiotic consumption. If we diagnose any STIs 
these will be treated in the normal way.           
 
 
 
                
     
We will then take the same additional samples as your first visit for the purpose of this study: 
• 
A urine sample 
• 
An anal swab 
• 
An oral rinse sample 
 
Know that you are also always welcome at ITM in between planned study visits in case of any 
questions, issues. 
You will not have to pay for any of the treatment and procedures performed specifically for this study. 
You will not receive any compensation for study participation. Your transport costs, if any, will also 
not be reimbursed. 


 
 
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RISKS AND INCONVENIENCES 
The risks related to this study for you are minimal. Participating in the study will mean that swabs are 
taken from your anus at both study visits; these swabs are however not invasive nor painful. 
Answering questions regarding your sexual behavior during study visits may be experienced as 
inconvenient.  
BENEFITS 
Your participation will give the scientific community an insight to the discovery of new ways to combat 
the resistance to antibiotics, which will likely become a major health problem in the near future. 
 
INSURANCE 
The organizer of this study, the Institute of Tropical Medicine, has obtained an insurance to cover any 
possible harm or injury that may be caused by participation in the study. If you get harmed or have 
questions about injuries as a result of being in the study, please contact the responsible researcher, 
Dr Chris Kenyon, 03 247 0786. 
You can find more detailed information in Part 3. 
 
STORAGE OF BODY SAMPLES 
We would like to store your collected samples (urine, anal swab, oral rinse) for a maximum of 20 years 
after study completion to be used for future scientific research related to sexually transmitted 
infection. Such future studies will be separately approved by the Ethics Committee in advance and will 
not involve any genetic testing of your own human DNA. 
 
You can indicate whether or not you agree with this future research in Part 2 of this document. Even 
if you decide that you do not want us to store your samples, you can still participate in the study. 
 
You can find more detailed information in Part 3. 
 
PRIVACY AND DATA PROTECTION 
We will do everything we can to protect your privacy. Information about you will be treated as strictly 
confidential; it will be stored in an electronic database, and identified by a code and not by your name. 
The documents where your name is mentioned will not be shared with anyone, except the study 
researchers, your doctor and few other people who have to keep it confidential, such as specific 
employees of the Institute of Tropical Medicine.  
All study data will be retained for a period of 20 years. The findings of the study will be published in 
(medical) journals. Your data can be shared with other researchers for future research, only upon 
condition that they are either pseudonymised (meaning that your name will be replaced by a code), 
or fully anonymized (meaning that the study code cannot be linked anymore to your name: nobody 
will know anymore that these data came from you).  
 
Your name will not appear in any database, report or publication resulting from this study.  
 
The study itself (and its results) will be published in the European public registry, 
http://www.clinicaltrialsregister.eu/, which is a public database of scientific studies with human 
participants. It does not contain any personal data of participants. 
 
In accordance with the European Data Protection Regulation (GDPR), you may request access to and 
correct your personal data if needed. 
 
You can find more detailed information in Part 3. 


 
 
ResistAZM – ICF Eng. Version 1.1, dated 3Sept2021 
 
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ETHICS COMMITTEE 
Before the start, this study was reviewed and approved by the Institutional Review Board of the 
Institute of Tropical Medicine in Antwerp and an independent ethical committee appointed by the 
Belgian regulatory authorities. Any changes to the conduct of the study will also need to be approved 
by these ethical review boards before they are implemented. 
 
VOLUNTARY PARTICIPATION 
Your participation in this study is entirely voluntary. It is your choice whether you want to take part in 
it or not. Whether you choose to participate or not, all the services you receive at this clinic will remain 
the same.  
 
You also have the right to stop your participation in the study at any time, even after you have signed 
this Informed Consent Form. You do not have to give a reason for stopping. 
Also the researcher can decide to stop your participation in this study at any moment as well, without 
asking your permission, if he/she judges this in your best interest, or if it appears that you do not 
follow the requirements for participation. Should this happen, the researcher will explain this to you, 
and discuss what will happen afterwards. 
 
CONTACT PERSON IN CASE OF QUESTIONS  
If you have any questions concerning your participation in this study, your rights or if you think you 
have been harmed as a result of the study, please contact, now, during, or after the study: 
 
 
Chris Kenyon  
Institute of Tropical Medicine 
Antwerp 
Tel 03 247 0786 (After hours: 03 821 3000. Ask for the Infectious Diseases doctor on-call) 
ckenyon@itg.be 
 
 
 
 
 
 


 
 
ResistAZM – ICF Eng. Version 1.1, dated 3Sept2021 
 
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Part 2. INFORMED CONSENT FORM 
CONFIDENTIAL 
ResistAZM study:  
An open label randomized controlled trial comparing the effect of ceftriaxone 
plus azithromycin versus ceftriaxone for the treatment of Neisseria 
gonorrhoeae on the resistome  
 
To be completed by the participant 
 
I have received a copy of the written participant information sheet (part 1). I also received verbally 
sufficient and understandable explanations, with enough time to ask questions, and my questions were 
satisfactorily answered. 
I freely consent to participate in this study and to cooperate in the study examinations/activities.  
I understand that during the study, my personal and medical data will be recorded, and that the 
confidentiality of this data will be protected according to the applicable laws in Europe and Belgium. 
 
 
 Yes / 
 No: I agree that samples collected for the study are stored for a maximum of 20 years after 
completion of this study and may be used for future scientific research related to sexually transmitted 
infections (not including human genetic testing). 
 
Date: __ __ / __ __ / __ __ __ __ 
 
Name of participant:          ____________________________________ 
 
Signature of participant:    ____________________________________ 
 
 
 
 
To be completed by the research staff obtaining the informed consent  
 
I, undersigned, ___________________________________ confirm that I have informed the participant 
about all the relevant aspects of this study. I confirm that he has consented voluntarily to participate in the 
study. 
 
Date: __ __ / __ __ / __ __ __ __ 
 
Signature:   _______________________________________ 
 
 
 
 


 
 
ResistAZM – ICF Eng. Version 1.1, dated 3Sept2021 
 
p. 6 of 8 
Part 3. ADDITIONAL INFORMATION 
 
ResistAZM study:  
An open label randomized controlled trial comparing the effect of ceftriaxone 
plus azithromycin versus ceftriaxone for the treatment of Neisseria 
gonorrhoeae on the resistome  
 
1. Insurance 
Any participation in a study involves a risk, however small it is. The sponsor is, even if there is no fault, 
responsible for damage caused to the participant (or in the event of death, his/her dependents) and 
directly or indirectly caused by his/her participation in the study. The sponsor has taken out insurance 
for this responsibility.   
You are therefore asked to report any new health problem to the investigator. He/she will be able to 
provide you with additional information concerning possible treatments. 
The insurance does not cover the natural progression of disease or known side effects of your normal 
treatment. If the investigator believes that a causal link between a new (health) complaint and your 
participation in the study is possible, he/she will inform the study sponsor, which will initiate the 
declaration procedure to the insurance company. The latter will appoint an expert - if considered 
necessary - to assess whether there is a link between your new health problems and the study. 
In the event of disagreement either with the investigator or with the expert appointed by the 
insurance company and also whenever you feel it is appropriate, you or - in case of death - your 
dependents may bring proceedings against the insurer directly in Belgium (AMLIN Insurance SE, B-
1030 Brussels, Koning Albert II-laan 37). 
The law provides that the insurer may be summoned to appear either before the judge of the location 
where the event giving rise to the damage occurred, or before the judge of your domicile, or before 
the judge of the insurer’s registered offices. 
 
2. What happens with your samples collected during the study  
Your biological samples collected for the purpose of the study are pseudonymised (i.e. your identity 
will be replaced by an ID code in the study) and thus they carry an identification code and not your 
name. 


 
 
ResistAZM – ICF Eng. Version 1.1, dated 3Sept2021 
 
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The Biobank Manager for the samples at the Institute of Tropical Medicine (Dr. Maartje Van 
Frankenhuijsen, Department of Clinical Sciences) undertakes to ensure that they will be used  within 
the context of clinical research and destroyed at the end of the scheduled storage period.  
If you withdraw your consent to take part in the study, you can also ask to have your samples 
destroyed. Contact the investigator or biobank@itg.be if you want to do so. The test results obtained 
from your samples before you withdraw your consent will however be retained by the study sponsor. 
Your samples will not be used for human genetic testing. Your samples will also not be sold to 
commercial companies. 
Possibly, your samples are interesting for future research which explains why they are preferably 
stored  for a longer period. To allow such a long term storage, your additional consent is requested in 
Part 2 of this document. This research will be in the same research domain or research about the same 
disease as in this study. It will also have to be approved by an Ethics Committee. Even if you agree with 
this secondary use of your samples, you have the right to have your samples destroyed at any later 
moment. You also have the right to have your stored samples anonymized1 at any moment. The long-
term storage and management of your samples always happens as described by the law2. 
The biological material collected within the study are considered as a “donation” and you should be 
aware that, in principle you will not receive any financial benefit (royalties) associated with the 
scientific research or the development of new diagnostics/therapies derived from the use of your 
donated samples. 
 
3. Data protection and rights of participants in the study  
Your participation in the study means that you agree that the investigator collects and records your 
personal and medical data, and that the study sponsor (that is the research institute that organized 
and conducts the study) uses them for research purposes and in connection with scientific and medical 
publications.  
Your data will be processed in accordance with the European General Data Protection Regulation 
(GDPR) and with the Belgian legislation on the protection of natural persons with regard to the 
processing of personal data. The sponsor of this study, that is Institute of Tropical Medicine, acts as 
data controller for your data and the lawful ground for the data processing is the public interest. 
You are entitled to ask the investigator what data are collected about you and how they are used for 
the study3. This data may concern your current (clinical) situation but also some of your demographic 
data, lifestyle data, (medical) background, the results of previous medical examinations , and obviously 
the results of examinations required by this protocol.  
 
1 Meaning that the link to your identifying information like your name is completely broken and thus cannot be traced back to you. 
2 Law on human body material of 2008 and the Royal Decree on biobanks of 2018. 
3 These rights are guaranteed by the European Data Protection Regulation 2016/679 (GDPR), the Belgian law on the protection of natural 
persons with regard to the processing of personal data of 30 July 2018 and the Law of 22 August 2002 on patient rights.  


 
 
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p. 8 of 8 
In case you wish to withdraw your consent for participation, the pseudonymised data that was 
collected about you before your withdrawal will be retained. This is to guarantee the validity of the 
research. 
The investigator has a duty of confidentiality with regard to the data collected. 
This means that he/she commits to never reveal your name in the context of a publication or 
conference, but also that he/she commits to pseudonymize your data, before they are recorded and 
processed for analysis. The investigator and his/her team will therefore be the only ones to be able to 
establish a link between the data recorded and analysed in the study and your name or medical 
records.  
Care will be taken not to transmit data that contain any combination of elements that might allow you 
to be identified4.    
To verify the quality of the study, it is possible that your study data will be examined by persons subject 
to professional secrecy and designated by sponsor of the study, the national regulatory authority,  or 
an independent audit body. In any event, this examination of your records may only take place under 
the responsibility of the investigator (or one of the collaborators designated by him/her).  
The sponsor will use the data collected within the context of the study in which you are taking part, 
but would also like to enable use of the data for future research in the same research context as this 
study. Any use of your data outside the context described in this document is only possible with the 
approval of an ethics committee and ITM’s data access committee. 
For future studies, your pseudonymised or anonymized data could also be transferred to countries 
outside Belgium and Europe where the standards about data protection may be different or less 
stringent. In such a case, the sponsor will assure that appropriate warranties are present with regard 
to the transfer and later use of the data in a secure and correct way. 
If you have any questions relating to how your data are being processed, you may contact the 
investigator. 
Also 
the 
data 
protection 
officer 
of 
ITM 
can 
be 
contacted 
(email: 
informatieveiligheid@itg.be or tel. 03 247 07 43). 
Finally, if you have a complaint concerning the processing of your data, you can contact the Belgian 
supervisory authority who ensures that privacy is respected when personal data are processed.  
Data Protection Authority (DPA) 
Drukpersstraat 35, 
1000 Brussels 
Tel. +32 2 274 48 00 
e-mail: contact@apd-gba.be 
Website: https://www.dataprotectionauthority.be  
 
 
 
4 The database containing the results of the study will therefore not contain any combination of elements such as your initials, your 
gender and your full date of birth (dd/mm/yyyy). 

Prot_000.pdf: 
 
 
1 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
 
ITM Clinical Trial ITM202101 
EudraCT 2021-003616-10 
NCT05027516 
 
An open label randomized controlled trial comparing the effect of ceftriaxone 
plus azithromycin versus ceftriaxone for the treatment of Neisseria 
gonorrhoeae on the resistome  
(ResistAZM Trial) 
 
Initial Protocol 
<Version 1.1, 3 September 2021> 
 
 
 
 
 
 
 
Sponsor: 
Institute of Tropical Medicine 
Nationalestraat 155 
B-2000 Antwerpen - Belgium 
Coordinating/Principal 
Investigator: 
 
Dr. Chris Kenyon 
 


2 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
 
Protocol Number 
ITM202101 
EudraCT Number 
2021-003616-10 
Study Title 
An open label randomized controlled trial comparing the effect of 
ceftriaxone plus azithromycin versus ceftriaxone for the treatment of 
Neisseria gonorrhoeae on the resistome 
Study Acronym 
ResistAZM 
Version 
V1.1 
Date 
 3 September 2021 
Study drugs 
Ceftriaxone 1g & Azithromycin 2g 
Funder 
Institute of Tropical Medicine, Antwerp, Belgium 
Sponsor 
Institute of Tropical Medicine, Antwerp, Belgium 
 
Sponsor study team 
Institute of Tropical Medicine, Antwerp, Belgium 
Name 
Function 
Contact details 
Dr. Chris Kenyon 
Coordinating/Principal 
Investigator 
ckenyon@itg.be 
Yven Van Herrewege 
Head of the CTU 
yvanherrewege@itg.be 
Emeline de Viron 
Clinical Research Scientist 
edeviron@itg.be 
Achilleas Tsoumanis 
Study Statistician 
atsoumanis@itg.be 
Harry van Loen 
Data Manager 
hvanloen@itg.be 
Diana Arango 
Data Reviewer 
darango@itg.be 
 
Clinical Study Team 
Institute of Tropical Medicine – HIV/STI clinic 
Name 
Function 
Contact details 
Dr. Chris Kenyon 
Coordinating/Principal 
Investigator 
ckenyon@itg.be 
Dr. Eric Florence 
Co-Investigator 
eflorence@itg.be 
Dr. Christophe Van Dijck 
Co-Investigator 
cvandijck@itg.be 
Dr. Tessa De Block 
Co-Investigator 
tdeblock@itg.be 
Dr. Said Abdelatti 
Co-Investigator 
sabdellati@itg.be 


3 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
Dr. Jolein Laumen 
Co-Investigator 
jlaumen@itg.be 
Dr. Thibaut Vanbaelen 
Co-Investigator 
tvanbaelen@itg.be 
Irith De Baetselier 
Laboratory coordinator 
idebaetselier@itg.be 
 
STATEMENT OF COMPLIANCE  
 
By signing this protocol, the Investigator(s) acknowledge(s) and agree(s): 
 
This protocol contains the necessary information for conducting this clinical study. The Principal 
Investigator will conduct this study as detailed herein and will make every reasonable effort to complete 
the study within the time designated. The Principal Investigator commits to carry out the study in 
compliance with the protocol, amendments, applicable procedures and other study-related documents 
provided by the Sponsor, and in compliance with the Declaration of Helsinki, Good Clinical [Laboratory] 
Practice (GC[L]P), the EU General Data Protection Regulation 2016/679 (GDPR), the ESF/ALLEA Code of 
Conduct for Research Integrity, and applicable regulatory requirements.  
The protocol and all relevant study information, which is provided by the Sponsor, will be made available 
to the physicians, nurses and other personnel who participate in conducting this study. The Investigator 
will use this material for their training so that they are fully informed regarding the drugs and the conduct 
of the study. 
The Sponsor of this study – the Institute of Tropical Medicine in Antwerp, Belgium (ITM) – can at any 
time have access to the source documents from which Case Report Form information may have been 
generated and will be permitted to perform trial-related monitoring and audits. All study material will be 
maintained according to regulatory requirements and until the Sponsor advises that retention is no longer 
necessary. 
 
 


4 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
 
COORDINATING/PRINCIPAL INVESTIGATOR: 
 
 
Title, Name: Prof. Dr. Chris Kenyon 
Date: 
03/09/2021 
 
Signed: 
 
 
 
 
 
 
 
 
 
 
 
DIRECTOR OF THE INSTITUTE OF TROPICAL MEDICINE: 
 
 
Title, Name: Prof. Dr. Marc-Alain WIDDOWSON 
Date: 
 
 
Signed: 
 
 
 
 
 
 
 
 
 
 
 
 
 
Signing this document, I commit to carry out the trial in accordance with the protocol, Good Clinical 
Practice and applicable ethical and regulatory requirements. I also acknowledge the paragraph 
relevant to study confidentiality and authorize the Institute of Tropical Medicine, Antwerp, Belgium to 
record my data on a computerized system containing all the data pertinent to the study. 
 
 
 
 
 
 


5 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
TABLE OF CONTENTS 
STATEMENT OF COMPLIANCE .......................................................................................................................... 3 
TABLE OF CONTENTS ........................................................................................................................................ 5 
SYNOPSIS ......................................................................................................................................................... 6 
1. INTRODUCTION ............................................................................................................................................ 8 
1.1 BACKGROUND AND RATIONALE ........................................................................................................................... 8 
2. STUDY OBJECTIVES ..................................................................................................................................... 15 
2.1 PRIMARY OBJECTIVE ........................................................................................................................................ 15 
2.2 SECONDARY OBJECTIVES ................................................................................................................................... 15 
3. STUDY DESIGN ............................................................................................................................................ 15 
3.1 GENERAL STUDY DESIGN ................................................................................................................................... 15 
4. PARTICIPANTS, POPULATION & SELECTION ................................................................................................ 17 
4.1 SETTINGS, SELECTION & RECRUITMENT ............................................................................................................... 17 
4.2 INCLUSION AND EXCLUSION CRITERIA .................................................................................................................. 17 
4.3 SAMPLE SIZE ................................................................................................................................................... 17 
4.4 RANDOMIZATION ............................................................................................................................................ 17 
4.5 WITHDRAWAL AND TERMINATION OF THE STUDY .................................................................................................. 18 
5. STUDY PROCEDURES .................................................................................................................................. 19 
5.1 STUDY/VISIT SCHEDULE .................................................................................................................................... 19 
5.2 OBTAINING INFORMED CONSENT ....................................................................................................................... 20 
5.3 SPECIFIC PROCEDURES AND ACTIVITIES ................................................................................................................ 21 
5.4 LABORATORY PROCEDURES ............................................................................................................................... 21 
6. STUDY INVESTIGATIONAL PRODUCT .......................................................................................................... 23 
6.1 PURCHASING, PREPARATION AND ADMINISTRATION .............................................................................................. 23 
6.2 PARTICIPANT COMPLIANCE MONITORING ............................................................................................................ 23 
6.3 PRIOR AND CONCOMITANT THERAPY .................................................................................................................. 23 
6.4 PACKAGING.................................................................................................................................................... 23 
6.5 RECEPTION, STORAGE, DISPENSING AND RETURN .................................................................................................. 24 
7. SAFETY ASSESSMENT .................................................................................................................................. 24 
7.1 (SERIOUS) ADVERSE EVENTS ............................................................................................................................. 24 
7.2 DATA AND SAFETY MONITORING BOARD ............................................................................................................ 26 
8. STATISTICAL METHODS .............................................................................................................................. 27 
8.1 STUDY HYPOTHESES AND HYPOTHESIS ................................................................................................................. 27 
8.2 VARIABLES OF INTEREST ................................................................................................................................... 27 
8.3 STATISTICAL METHODS ..................................................................................................................................... 27 
8.4 SAMPLE SIZE AND POWER ................................................................................................................................. 28 
9. MONITORING AND QUALITY ASSURANCE .................................................................................................. 29 
10. DATA MANAGEMENT ............................................................................................................................... 29 
11. ETHICAL ISSUES ........................................................................................................................................ 31 
11.1 ETHICAL AND REGULATORY REVIEW .................................................................................................................. 31 
11.2 PROTOCOL AMENDMENTS .............................................................................................................................. 31 
11.3 INFORMED CONSENT ...................................................................................................................................... 31 
11.4 CONFIDENTIALITY .......................................................................................................................................... 32 
11.5 RISKS AND BENEFITS ...................................................................................................................................... 32 
11.6 COMPENSATION FOR PARTICIPATION ................................................................................................................ 32 
11.7 INSURANCE .................................................................................................................................................. 32 
12. DISSEMINATION OF RESULTS, INTELLECTUAL PROPERTY .......................................................................... 32 
13. ARCHIVING ............................................................................................................................................... 33 
14. REFERENCES ............................................................................................................................................. 33 
15. LIST OF ABBREVIATIONS ........................................................................................................................... 38 




7 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
HYPOTHESIS 
CRO/AZM is more resistogenic than CRO 
DESIGN 
Unblinded, single center, randomized controlled 
trial 
STUDY SITE & POPULATION  
 
Subjects will be recruited from the HIV/STI Clinic 
at the Institute of Tropical Medicine Antwerp. A 
maximum of 42 will be recruited (21 in each arm) 
DURATION 
Each participant will be enrolled for 15 days. 
Total study duration will be 8 months 
OBJECTIVES AND ENDPOINTS 
 
Primary Objective: Assess if there is a difference in 
the ratio of macrolide resistance determinants in the 
anorectal microbiome of the CRO/AZM group to 
those in the CRO group at 14 days post-treatment.        
Primary Endpoint: The mean macrolide resistance 
determinants in the day 14 anorectal samples in the 
CRO/AZM group over the corresponding mean 
quantity in the CRO group 
INCLUSION & EXCLUSION CRITERIA 
 
• 
Inclusion criteria: 
1. Able and willing to provide written informed 
consent 
2. Male sex at birth 
3. At least 18 years old 
4. Confirmed diagnosis of urethritis, proctitis or 
pharyngitis Ng – symptomatic or asymptomatic  
• 
Exclusion criteria: 
1. Use of any macrolide antibiotics in previous 6 
months 
2. Known contra-indications or allergy to 
ceftriaxone, azithromycin or lidocaine                              
3. Indication for any other antibiotic treatment at 
the time of enrollment 
SCREENING, RECRUITMENT & RANDOMIZATION 
 
Subjects (identified from the HIV/STI clinic) will 
be screened at ITM. If eligible, they will be 
enrolled and randomized to group 1 (CRO) or 
group 2 (CRO/AZM) 
STUDY DRUG 
 
Group 1:  Single dose of Rocephine® (= 
ceftriaxone 1g + lidocaine 35mg) intramuscular 
injection  
Group 2:  Single dose of Rocephine® (= 
ceftriaxone 1g + lidocaine 35mg) intramuscular 
injection + single dose of azithromycin 2g orally 
FOLLOW-UP 
Single follow-up visit at day 14 
SAFETY 
 
Safety of the intervention will be evaluated by 
recording (Serious) Adverse Events. Data will be 
monitored by means of a sponsor regulated 
pharmacovigilance system 
STATISTICAL METHODS 
The primary analysis of assessing a difference 
between 
the 
mean 
macrolide 
resistance 
determinants in anorectal microbiome between the 
two groups will be done using permutation test.  
 


8 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
1.  INTRODUCTION 
 
1.1 Background and Rationale 
 
Neisseria gonorrhoeae (Ng) has developed resistance to all antimicrobials used to treat it. As a result, 
there are fears that it may become untreatable in the near future [1-3]. Although Ng has been shown 
to use the full repertoire of possible resistance mechanisms [4], it is particularly adept at taking up 
genetic material from its environment via a well-developed system of transformation [5, 6].   
Phylogenetic analyses have, for example, provided strong evidence that Ng acquired the cefixime-
resistance-inducing PBP2A gene from commensal pharyngeal Neisseria [7]. The same has been 
established with macrolide resistance ￼8-11]. A range of studies have concluded that excessive 
macrolide consumption first results in macrolide resistance in commensal Neisseria and that this 
resistance is then transferred to Ng via transformation ￼8-11]. An important implication of Ng’s 
ability to take up resistance mechanisms from other bacteria is that it can acquire resistance by 
passing through environments that have been enriched with relevant resistance genes (Figure 2) [12].  
 
Figure 2. A schematic illustration of how the AZM (azithromycin) component of ceftriaxone 
/azithromycin (CRO/AZM) dual therapy could result in AZM resistance in Ng (Neisseria gonorrhoeae). 
AZM given at day 0 results in a prolonged increase in the proportion of commensal oral 
Neisseria/Streptococci that have macrolide resistance (blue squares). A new Ng infection during this 
period of elevated resistance could acquire the resistance conferring determinants from these resident 
commensals via horizontal gene transfer. 
 
The importance of evaluating the effect of antibiotics on the resistome 
Importantly this means that AMR surveillance needs to monitor the prevalence of AMR determinants 
in commensal Neisseria and other bacteria and not just pathogens such as Ng. The commensal 
Neisseria can be viewed as a canary-in-the-mine type of early warning system [13]. In particular when 
evaluating the risks and benefits of various antimicrobial options, we need to evaluate their effects 
CRO/
AZM
New Ng 
infection
Acquisition of 
AZM resistance
Resistant 
NG


9 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
on the commensal resistome. The primary objective of this study is to compare the effects of 
ceftriaxone (CRO) vs. ceftriaxone/azithromycin (CRO/AZM) on the resistome.  
 
Switch in treatment guidelines and absence of clinical evidence of superiority of CRO/AZM vs CRO 
in the treatment of N. gonorrhoeae 
There are currently two main schools of thought as to the optimal therapy of Ng: monotherapy with 
CRO and dual therapy with CRO/AZM (henceforth termed dual therapy). Dual therapy rose to 
prominence in multiple countries around 2012 [14, 15]. Around this time both the European IUSTI and 
the United States CDC Ng treatment guidelines changed to advocating dual therapy [14, 15]. The 
rationale was not based on any clinical trials or observational studies, but on expert opinion that dual 
therapy would be predicted to retard the emergence of gonococcal AMR. Of note the combination of 
AZM and CRO has not been shown to exhibit any synergy in vitro or in vivo [16, 17]. No RCTs have 
been performed to directly compare the two treatment options (Box 1 – page 12) [18]. Over the past 
2 years a number of prominent groups have reevaluated the evidence for the two treatments options 
and changed their recommendations. In the case of the United States and the United Kingdom, the 
national guidelines have recently been changed to explicitly recommend monotherapy with CRO as 
the preferred therapy for Ng [19]. The European IUSTI Ng treatment guidelines were also revised in 
2020 to include mono-therapy as an alternative treatment strategy (Box 1). The guidelines in the 
Netherlands continue to recommend CRO monotherapy [20].  
 
Five lines of evidence contributed to this shift: 
1. Increasing AZM resistance. In Belgium the percentage of Ng isolates with reduced 
susceptibility to AZM (MIC – Minimum Inhibitory Concentration > 1mg/L) has increased from 
0.2 to 16% over the past 8 years – the period that dual therapy has been used [21]. An increase 
in AZM resistance has been noted in multiple countries in Europe [22]. In the USA there has 
been an increase in AZM reduced susceptibility isolates from 0.6% in 2013 to 4.6% in 2018 
[19]. The prevalence of Ng AZM resistance is so high in a number of populations around the 
world that certain authors have argued that its use can no longer be justified [15, 24, 25]. 
Confirmed cases of treatment failure following dual therapy have reinforced this perspective 
[26]. 
2. Macrolide resistance and stewardship. AZM is classified by the WHO AWARE typology as a 
second line antimicrobial whose use should be avoided where ever possible so as to prevent 
the emergence of AMR and preserve its clinical utility [27, 28]. The use of dual therapy for Ng 
has played an important role in elevating macrolide consumption levels in contemporary HIV 
Pre-Exposure Prophylaxis (PrEP) populations to 7-fold higher than thresholds for the induction 
of macrolide resistance in a range of bacteria including Treponema pallidum, Mycoplasma 
genitalium, Streptococcus pneumoniae and Ng [21, 29, 30]. We have confirmed this high 
macrolide consumption both in Belgium and other countries and shown that treatment for 
Ng plays an important role in driving this high consumption [21, 31]. In Belgium we have gone 
one step further and ascertained that the prevalence of high level resistance to AZM in 
commensal Neisseria species in Men who have Sex with Men (MSM) attending the ITM STI 
clinic is considerably higher than that ever recorded anywhere in the world [32]. We have 
hypothesized that a likely driver of this resistance is high levels of macrolide consumption 
including those used as dual therapy for Ng [32].  
 
These concerns have been reinforced by studies that have demonstrated the long-term adverse 
effects of macrolides on the resistome. Studies have shown that the quantity of ermB genes 
(conferring macrolide resistance) in the colonic microbiome for example increase 3 to 5 orders of 


10 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
magnitude for 2 to 4 years after a single course of macrolides [33-35]. These changes in the resistome 
are mediated by increases in macrolide-resistant enterococci [36], streptococci [37] and staphylococci 
[36, 38].  A more recent study, MORDOR, found that 6-monthly mass administration of AZM to 
children in Niger, resulted in a large increase in the abundance of genes in the gastro-intestinal tract 
conferring resistance to macrolides and  a range of other classes of antimicrobials (Figure 3) [39]. This 
study also revealed a significant increase in the prevalence of macrolide resistance in S. pneumoniae 
in communities receiving mass AZM [40]. Other studies have found that a single course of AZM can 
lead to elevated AZM MICs in oropharyngeal streptococci and that this effect can persist for 6 months 
[37].     
 
Figure 3. Results from the MORDOR study showing the difference in abundance of antibiotic-resistance 
determinants in the azithromycin-treated group as compared with the group that received placebo, 
with associated 95% confidence intervals. The abundance of macrolide and other antibiotic classes 
resistance determinants was ascertained from rectal swabs from recipients at 36 months and 48 
months (From [39]). 
 
 
3. Pharmacokinetic considerations of dual vs. monotherapy. Although human studies have not 
been performed to evaluate the length of time that CRO needs to be above the MIC to 
eradicate Ng at each relevant anatomical site, Monte Carlo models have revealed that this 
time should be above 20 hours to eradicate urogenital Ng [41]. Various lines of evidence 
including those from a mouse model of Ng suggest that a CRO dose of 500-1000mg given IM 
will provide a time above the MIC of 24 hours which is sufficient to eradicate Ng [42]. These 
considerations suggest that monotherapy with CRO dosed at 500mg to 1g IM would be 
sufficient to consistently eradicate Ng. 
 
4. Epidemiology of Ng in populations using monotherapy. Certain countries such as the 
Netherlands never advocated the use of dual AZM/CRO therapy in their national guidelines. 
There is no evidence of either a higher treatment failure rate or a decrease in cephalosporin 
and other antimicrobial class susceptibility in these populations [18].  
 
5. Importance of horizontal versus vertical transmission of AMR. The rationale for using 
multiple agents to prevent the emergence of AMR is likely to be stronger in infections where 


11 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
horizontal gene transfer (HGT) does not play a prominent role such as Mycobacterium 
tuberculosis (MTB) and Plasmodium falciparum [31, 43, 44]. In the case of MTB, using 
rifampicin in combination with isoniazid has been shown to reduce the probability of 
resistance emerging to either agent. Combination therapy will not result in isoniazid 
resistance via selection of resistance in commensal Mycobacteria and subsequent 
transmission of this resistance to MTB via HGT [44]. This is because HGT occurs at an 
extremely low frequency in MTB [44]. 
 
There is however increasing evidence that this indirect collateral pathway plays an important 
role in the emergence of AMR in Ng [7, 8, 31, 43]. Using AZM in dual therapy may thus select 
for AMR in Ng via first selection of macrolide resistance in commensal Neisseria or other 
bacteria (Figure 2) [8, 9]. As noted already a range of studies have confirmed that macrolide 
resistance in Ng has emerged via this pathway. Wadsworth et al., for example have 
established that the acquisition of sections of the mtrCDE gene by Ng from commensal 
Neisseria has played a crucial role in the genesis of macrolide resistance in Ng [8, 9]. 
 
We have found evidence that macrolide resistance determinants may be transferred from oral 
streptococci to commensal Neisseria and play an important role in macrolide resistance. In a 
whole genome analysis of the determinants of high-level AZM resistance in circulating 
Neisseria subflava in MSM attending the ITM STI clinic, for example we found clear evidence 
that 9 out of 11 isolates had taken up the msrD gene from oral streptococci and that this in 
turn was the major determinant of high level AZM resistance (MICs > 256 mg/L; unpublished 
data).  
 
These five types of evidence have been instrumental in effecting the change to CRO monotherapy in 
the USA and UK treatment guidelines and the European IUSTI guidelines newly classifying CRO 
monotherapy as alternative therapy. The fact that there is this still discrepancy in treatment 
guidelines, taken in conjunction with the absence of RCTs comparing these different treatment 
options could be reasonably interpreted as reflecting equipoise in the choice between mono- and 
dual-therapy. In this setting, there is a crucial need to generate better evidence to inform guidelines. 
Conducting large RCTs to compare the clinical efficacy of mono- versus dual-therapy would be costly. 
As an alternative/complementary strategy, in this study we will conduct a small RCT with as primary 
outcome the difference in the abundance of resistance conferring genes in the rectal microbiome in 
the two arms 14 days after the receipt of therapy.  
 
The specific hypothesis we will be testing is that dual therapy increases the probability of the 
acquisition of macrolide resistance by Ng indirectly via its effect on commensal organisms. This is 
illustrated in Figure 2. The Y axis represents the proportion of oral Streptococci/Neisseria that are 
macrolide resistant following the receipt of AZM/CRO. The figure is based on an RCT by Malhotra-
Kumar et al., who administered 3 days of AZM or placebo to healthy volunteers [37]. The study found 
a large increase in the proportion of oral streptococci that exhibited macrolide resistance between 
days 4 to 28. In a small study conducted in MSM attending the STI clinic at ITM, we have found that 
the receipt of CRO/AZM for Ng is associated with an increase in the proportion of all Neisseria isolates 
that have AZM resistance 14 days following treatment [32]. This was in a very small study without a 
control group. Based on this work, and other lines of evidence, we hypothesize that the receipt of 
CRO/AZM will result in a greater increase in macrolide resistance in commensal Neisseria and other 
bacteria in both the pharyngeal and rectal microbiomes than CRO. Furthermore, we hypothesize that 
this effect will persist for at least 14 days and that incoming Ng could acquire AZM-resistance-
conferring-determinants from these commensals.  
 




13 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
Using an oropharyngeal rinse to characterize the microbiome, resistome of the oropharynx 
Our research team as well as others have been trying to optimize a protocol to accurately characterize 
the microbiome and resistome of the oropharynx. Initially we collected up to 3 swabs to do this [46-
48]. This protocol had numerous problems including being laborious, unpopular with participants and 
subject to interindividual sampling errors. More recently we have had good results using a 
oropharyngeal rinse specimen (henceforth termed an oral rinse).  
This methodology was developed and validated by Caselli et al., who found that an oral rinse specimen 
provided an effective way to characterize the oropharyngeal microbiome [49]. An oral rinse is also less 
invasive/uncomfortable than taking a posterior pharyngeal/tonsillar swab [49]. The oral rinse 
specimen was particularly good at characterizing the oral Neisseria species, which is the main focus of 
our research. The oral rinse technique is easy and quick to perform. It involves the individual rinsing 
their mouth with 15ml of sterile phosphate buffered saline (PBS) for 60 seconds and then spitting this 
out into a container.   
 
Morbidostat: an alternative method to assess if CRO/AZM could be more resistogenic than CRO 
One of the secondary objectives of the study will be to use the morbidostat to test if dual therapy is 
more likely to transfer macrolide resistance to Ng than monotherapy. The morbidostat is an in-vitro 
culture system that is able to place Ng under varying antimicrobial selection pressures [50]. We have 
previously used it as a way to map out the molecular pathways to high level macrolide and 
chlorhexidine resistance [50, 51]. In this experiment we will compare the time to the emergence of 
high-level macrolide resistance between the two treatment arms in Ng cultured in the morbidostat 
following regular infusions of DNA extracts of commensal Neisseria obtained from the day 14 swabs 
of participants (Figure. 3). The hypothesis we will be testing is that the commensal organisms obtained 
from the dual therapy arm are more likely to contain DNA that confers resistance to macrolides and 
this DNA can be taken up by Ng and accelerate the acquisition of AZM resistance. 
 




15 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
2. STUDY OBJECTIVES 
 
2.1 Primary objective 
Assess if there is a difference in the mean macrolide resistance determinants in the anorectal 
microbiome of the CRO/AZM group compared to those in the CRO group at 14 days post-treatment.   
  
2.2 Secondary objectives 
• 
Assess if there is a difference in the mean non-macrolide1 resistance determinants in the 
anorectal microbiome of the CRO/AZM group compared to those in the CRO group at 14 days 
post-treatment.  
• 
Assess if there is a difference in the mean macrolide and non-macrolide1 resistance 
determinants in the oropharyngeal microbiome of the CRO/AZM group to those in the CRO 
group at 14 days post-treatment.  
• 
Assess if there is a difference in the proportion of oropharyngeal Neisseria that are macrolide 
resistant in the CRO/AZM group compared to the CRO group at day 0 and day 14 and the 
CRO/AZM group at day 0 
• 
Assess if the DNA extracts from the oropharyngeal Neisseria cultures obtained from the 
CRO/AZM group at day 14 result in a more rapid acquisition in AZM resistance than those from 
the CRO group 
• 
 Assess if the oropharyngeal commensal Neisseria and commensal Streptococci obtained from 
the CRO/AZM group at day 14 have less of an inhibitory effect on the growth of Ng than those 
obtained from the CRO group 
 
 
3. STUDY DESIGN 
3.1 General study design 
This is an open label, single center, randomized controlled trial in MSM attending the ITM HIV/STI 
clinic. A total of 42 subjects will be recruited and randomized (1:1) to one of the following groups: 
1) CRO/AZM (n= 21) 
2) CRO (n= 21) 
 
Visit 1 Day 0 
Diagnosis of Ng will be by a positive Nucleic Acid Amplification Test (NAAT) performed according to 
ITM’s current laboratory protocols or for patients with urethritis, a positive gram/methylene blue 
stain or culture result. Details of Ng infection will be collected (site of diagnosis, symptomatic, 
methods of confirmation and date). 
After the informed consent procedure, urine - oral rinse and anorectal swab samples will be taken, for 
Ng culture (of the site where Ng was detected), for microbiome/resistome profiling, and culturing 
oropharyngeal streptococcal and Neisseria species (Table 1).    
 
 
                      
Further STI screening will be in accordance with current local best practice and in consultation with 
 
1 aminoglycosides, bacitracin, beta-lactams, cationic peptides, elfamycins, fluoroquinolones, metronidazole, rifampin, 
sulphonamides, tetracyclines, trimethoprim 


16 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
the patient. Generally, for MSM, this includes a test for syphilis, HIV and hepatitis C unless these were 
carried out recently. All diagnosed STIs will be managed according to local best practice.  
Participants will be randomized 1:1 to either an intramuscular injection of CRO 1g or an intramuscular 
injection of CRO 1g plus a 2g oral dose of AZM. 
As it is recommended by IUSTI (International Union Against Sexually Transmitted Infections) Europe 
gonorrhoea treatment guidelines, participants will be asked to return to clinic 14 days after their 
treatment for a follow-up visit [14][59, 60]. Other aspects of routine STI care such as partner tracing 
will be carried out in accordance with current best practice guidance [14]. 
Visit 2 Day 14 
During this visit, swabs from the previously infected sites will be taken for NAAT and culture to assess 
clearance of Ng in line with current guidelines [14, 59]. Oral rinse and anorectal swab will be taken for 
microbiome/resistome profiling and DNA extracts for transformation studies. If it is not possible for 
the participant to return at day 14, then they will be asked to return on day 13 or 15.   
In addition, participants will be asked to return to the clinic at any point if they develop symptoms or 
signs of gonococcal infection. If any results reveal that the participant has an ongoing or new Ng 
infection further specimens will be taken for culture, sensitivity and phylogenetic analyses. The 
participants will also be advised to return for retesting for STIs at 12 weeks in accordance with current 
best practice guidelines [14]. This visit will not be included as a study visit. 
 
Figure 6. Study flowchart                                                                                                                                                                   
Legend: CRO: ceftriaxone; AZM: Azithromycin 
 
 
 
 
 
 
 
Screening
Enrollment & 
Randomization 1:1
CRO
CRO/AZM
CASI/Swabs
CASI/Swabs
Day 0
Day 14
Swabs 
Swabs 


17 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
4. PARTICIPANTS, POPULATION & SELECTION 
4.1 Settings, selection & recruitment 
This study will be performed in MSM attending the HIV/STI Outpatient Clinic of the ITM. MSM with a 
diagnosis of symptomatic or asymptomatic urethritis, proctitis or pharyngitis Ng will be invited for a 
screening visit by a member of the site research team. 
During the screening visit, further inclusion and exclusion criteria will be evaluated. 
The first act of recruitment will be considered as the moment the study doctors inform the HIV/STI 
physicians that potentially eligible subjects can be referred to them for screening.  
 
4.2 Inclusion and exclusion criteria 
The inclusion and exclusion criteria for this study will be as follows: 
Inclusion criteria: 
1. Able and willing to provide written informed consent 
2. Male sex at birth 
3. At least 18 years old 
4. Confirmed diagnosis of urethritis, proctitis or pharyngitis Ng – symptomatic or asymptomatic 
(Diagnosis of Ng will be by a positive NAAT performed according to the ITMs current laboratory 
protocols or for patients with urethritis a positive gram/methylene blue stain) 
Exclusion criteria: 
1. Use of any macrolide antibiotics (azithromycin, clarithromycin, erythromycin, 
roxithromycin, spiramycin) in previous 6 months 
2. Known contra-indications or allergy to ceftriaxone, azithromycin or lidocaine 
 
3. Presence of any other condition, including other STIs that will (likely) require the 
administration of another antibiotic at the time of enrollment, as assessed by the treating 
physician 
 
4.3 Sample size 
A total of 42 subjects will be enrolled in the study (21 subjects in each arm). This includes a drop-out 
rate of 5%. Detailed calculations of the sample size can be found in the statistical section 8.4. 
 
4.4 Randomization 
After all applicable screening assessments have been performed, subjects who have met all inclusion 
criteria and none of the exclusion criteria will be randomized (1:1) to one of the following groups and 
will receive a unique randomization number (Unblinded – Open Label study): 
 
1) CRO/AZM (n= 21) 
2) CRO (n= 21) 
 
The randomization schedules will be prepared by an independent sponsor biostatistician. The overview 
of the randomization list will not be shared with the investigators until the trial database is locked. The 
randomization list will be prepared using SAS 9.4 (SAS Institute, Cary NC). 
 


18 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
4.5 Withdrawal and termination of the study 
Reasons for Withdrawal 
In accordance with the Declaration of Helsinki, ICH Good Clinical Practice Guidelines and the Belgian 
Law on experiments of 2004, a participant has the right to withdraw from the study at any time for any 
reason without prejudice to his/her future medical care by the physician at the institution. The 
Investigator also has the right to withdraw patients from the study if one or more of the following 
events occur:  
• 
The participant withdraws the consent 
• 
The Investigator judges that further participation would have negative effect on the 
participant’s health.  
Handling of Withdrawals 
A complete final evaluation should be made at the time of the patient’s withdrawal. The Study Status 
Outcome form in the case report form should be completed with an explanation of why the patient is 
withdrawing. 
Participants withdrawn from study will continue to receive standard of care for their condition. This 
includes, if needed, care of any adverse event or complication, whether related or not to the study 
procedures. The Principal Investigator (PI) will assure this standard of care is provided.  
Participants will be considered lost to follow-up at study closure if they discontinued study visits 
without informing the study staff and could not be traced. When the investigator has no news of the 
participant, they must make every effort to contact him (at a minimum telephone the participant three 
times and send an email to them), to establish the reason for the discontinuation of treatment, and to 
suggest the participant comes to an end-of-study visit. If all these attempts to contact the participant 
fail, the investigator can then declare the participant “lost to follow-up”. The investigator should 
document all these attempts in the corresponding medical file. 
Participants informing the study staff about their withdrawal from trial will be considered as early 
withdrawals. 
Participants have the right to withdraw their consent at any time and to ask for retrospective 
withdrawal of all personal data/samples related to the trial. 
Termination of Study  
The study may be prematurely closed or interrupted by the sponsor in case of futility or adverse health 
outcomes for the study participants.   
If the study is prematurely terminated or suspended, the Sponsor will promptly inform the 
investigators/institutions, and the regulatory authority of the termination or suspension and the 
reason(s) for the termination or suspension.  The EC’s will also be informed promptly and provided the 
reason(s) for the termination or suspension by the Sponsor or by the investigator/institution, as 
specified by the applicable regulatory requirement(s). 
 
 
 
 
 
 
 
 


19 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
5. STUDY PROCEDURES 
5.1 Study/visit schedule 
 
Visit 1 Day 0 = Screening and Enrollment visit: 
- 
Check inclusion and exclusion criteria (Eligibility)  
- 
Informed Consent Process 
- 
Behavioral questionnaire  
- 
Review STI history, HIV serostatus, use of PrEP (prior 12 months) 
- 
Record previous antibiotic use (prior 12 months) 
- 
Oral examination 
- 
Physical examination (as indicated clinically) 
- 
Randomization  
- 
Sample collection for the following laboratory procedures:  
o First-void urine and anorectal swab for Ng culture 
o Ng culture will be performed from all the sites where the Ng was detected before 
study enrolment  
o STI screening as required  
o Oral rinse sample for microbiome/resistome analysis and for Ng culture 
o One additional anorectal swab for microbiome/resistome analysis 
- 
Dispensing and administration of CRO and AZM (according to CRO vs CRO/AZM arms) 
 
Visit 2 Day 14 (range 13-15 days): 
- 
Behavioral questionnaire  
- 
Record antibiotic use 
- 
Record (Serious) Adverse Events ((S)AE) 
- 
Physical examination (as indicated clinically) 
- 
Sample collection for the following laboratory procedures:  
o First-void urine and anorectal swab for Ng culture and for Molecular Ng testing 
o Ng culture will be performed from all the sites where the Ng was detected before 
study enrolment 
o Oral rinse sample for microbiome/resistome analysis and for Ng culture 
o One additional anorectal swab for microbiome/resistome analysis 
 
Unscheduled Visits:  
As it is currently the case for all STI clients, participants of this study will be able to attend the ITM 
clinic at any point in between the scheduled visits for any health-related concerns. Participants will be 
encouraged to attend the clinic for any symptoms compatible with an STI. 
 
A schematic overview of all study assessments can be found below in Table 1. 
 
Costs and reimbursements 
Patients will be provided the antimicrobials free of charge. The patients will not be charged for any 
tests beyond those that are routinely conducted for patients.    
 
Procedures and assessments 
A summary of the assessments in the trial are shown in Table 1 below. 
 
 
 
 


20 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
Table 1. Schedule of Assessments 
 
Visit 1 Day 0 
Visit 2 Day 14 
Eligibility 
x 
 
Details of Ng infection  
- site of diagnosis, symptomatic, methods 
of confirmation, date 
x 
 
Informed Consent 
x 
 
STI history, HIV serostatus, use of PrEP  
x 
 
Antibiotic use  
x 
x 
Behavioral questionnaire   
x 
x 
Oral examination 
x 
 
Physical examination  
- if clinically indicated 
x 
x 
(Serious) Adverse Event collection 
 
x 
Randomization 
x 
 
IMP dispensing & administration 
x 
 
Urine  
(Molecular Ng testing and/or Ng culture) 
x 
x 
Anorectal swab 1 
(Molecular Ng testing and/or Ng culture) 
x 
x 
Anorectal swab 2 
(Microbiome/resistome analysis) 
x 
x 
Oral rinse 
(Microbiome/resistome analysis) 
(Molecular Ng testing and/or Ng culture) 
x 
x 
Ng culture of the site of infection 
- 
includes 
assessment 
of 
antimicrobial 
susceptibility 
x 
x 
 
Legend: Ng testing: Neisseria gonorrhoeae testing ; STI: Sexually Transmitted Infection 
 
5.2 Obtaining informed consent 
The Informed Consent Form (ICF) documents will be designed in accordance with the requirements of 
the Helsinki Declaration (2013), the E6 ICH GCP Guidelines (2016) and the Belgian Law on Experiment 
on the Human Person (2004). The ICFs will be developed in Dutch, French and English. The 
translation(s) will be reviewed and translation validation forms will be completed and signed by both 
the translator(s) and reviewer(s).  
The ITM SOP for ‘Obtaining the Informed Consent of clinical trials’ subjects will be followed. The IC 
procedure will describe the purpose of the study, the procedures to be followed, the risks and benefits 
of participation, confidentiality issues, etc.  All informed consent procedures will be conducted during 
the Screening and Enrolment Visit by qualified staff members identified by the PI and done in the 
language chosen by the participant. Participants will be informed that participation in the study is 
completely voluntary and that they may withdraw from the study at any time without any negative 
consequences. Participant Information Sheets and consent forms will be provided to the study 
participants for review. The participants will be given enough time to consider whether or not to 
participate in the study. Upon agreement on participation, the consent form will be signed in two 
copies, namely by the participant and by the investigator administering the consent. The participant 


21 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
will receive one copy of the ICF, while the other copy will remain in the Investigator file. If a participant 
is unable to read or write, a signature from a witness to the informed consent discussion will be 
obtained.  
No participant may be enrolled in the study and no study sample collection will take place until the 
study investigator or designee has obtained his informed consent.  
Participants will be asked orally and in writing on the ICF to consent for long term storage of samples 
pertaining to the study (20 years after final study report) for future testing, with the exception of 
human genetic testing. As noted in the ICF, any genetic testing will only be directed to the microbiome 
and not the human genome. Future testing may include repeat and confirmatory testing to arrive at 
a proper diagnosis, testing that is already specified in this protocol but will be done on batched 
samples after all samples have been collected, or additional research. Concerning future additional 
researches, there is a very small likelihood of incidental findings. Before samples will be used, approval 
from the sponsor and the ethics committee will need to be obtained. If a participant refused long-
term storage, his samples will be destroyed after the submission of the final study report. Samples 
will be registered in the ITM biobank. 
 
5.3 Specific procedures and activities 
The following study specific activities will be conducted at each study visit:  
 
Physical examination:  
• 
Oral health assessment by study physician 
• 
Physical examinations as indicated clinically 
 
Collection of swab: 
The study physician or participant will take the anorectal swab according to clinical SOPs 
 
Collection of rinse: 
The physician will provide detailed instructions to the participant as to how to take the oral rinse 
sample and will supervise this specimen being taken 
 
Collection of first-void urine sample (collected by the participant) 
  
Behavioral Questionnaire: Visit 1 Day 0 includes antibiotic use in last year and Visit 2 Day 14 includes 
AZM/CRO side effects 
 
5.4 Laboratory procedures 
All laboratory procedures except for microbiome and resistome testing will be performed at ITM.  
Please refer to section 5.1 Study/Visit schedule when samples are taken and below-mentioned assays 
are performed.  
5.4.1. Laboratory testing performed at ITM 
Laboratory testing at ITM is always performed according to their validated SOPs. How samples will be 
collected, processed, stored, analyzed and destroyed will be documented in the laboratory analytical 
plan. 
Testing at the Clinical Reference Laboratory (CRL)  
- 
Ng molecular testing will be performed at the STI Reference Laboratory on urine, 
oropharyngeal and anorectal samples.  
- 
Culture of commensal oropharyngeal Neisseria and Streptococcal species 
- 
Culture of Neisseria gonorrhoeae in case Ng was detected using standard techniques  


22 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
The oral rinse, urine, anorectal, and swabs will be stored at -20°C until assayed. Microbial DNA will be 
extracted as per the current laboratory protocol from the oral rinse and anorectal swab separately 
according to standard laboratory procedures [61].   
Morbidostat: 
assessing 
if 
CRO/AZM 
could 
be 
more 
resistogenic 
than 
CRO                             
The morbidostat will be used to test if dual therapy is more likely to transfer macrolide resistance to 
Ng than monotherapy [50, 51]. In this experiment we will compare the time to the emergence of low- 
and high-level macrolide resistance (MIC defined as >1mg/L and >256mg/L, respectively) between the 
two treatment arms in Ng cultured in the morbidostat following regular infusions of DNA extracts of 
commensal Neisseria obtained from the day 14 swabs of participants. The hypothesis we will be testing 
is that the commensal organisms obtained from the dual therapy arm are more likely to contain DNA 
that confers resistance to macrolides and that this DNA can be taken up by Ng and accelerate the 
acquisition of AZM resistance. The Ng morbidostat has 15 vials and they will be divided into the 
following groups: (1) adding day 14 DNA extracts from the cultured commensal Neisseria species. from 
the CRO/AZM group (3 vials); (2) adding day 14 DNA extracts from the cultured commensal Neisseria 
spp. from the CRO group (3 vials); (3) adding day 0 DNA extracts from the cultured commensal 
Neisseria spp. from the CRO/AZM group (3 vials); (4) adding day 0 DNA extracts from the cultured 
commensal Neisseria species. from the CRO group (3 vials); (5) adding no DNA (3 vials). All experiments 
will be performed with the WHO-F strain of Ng. The time to the development of AMR is defined as the 
time between the start of the experiment and the time that this resistance threshold is first crossed. 
All vials will be placed under an AZM selection pressure as described elsewhere [51]. 
Evaluating 
the 
effect 
of 
CRO 
vs. 
CRO/AZM 
on 
colonization 
resistance                             
We will assess if CRO/AZM diminishes the prevalence/abundance of inhibitory Neisseria and 
Streptococcal commensals to a greater extent than CRO. To test this hypothesis, we will use agar well 
diffusion assays to compare the inhibitory zone produced by oropharyngeal microbiota after receipt 
of either CRO/AZM or CRO. 
In pilot experiments using agar overlay and agar well diffusion assays, we found that the oropharyngeal 
microbiota from certain individuals in the PReGo study showed strong inhibition of Ng whereas that 
from others had little or no inhibitory effect. We have found the agar well diffusion method to be 
easier to perform and the inhibitory effect to be easier to interpret via automated methods.  
We will compare the colonization resistance following receipt of CRO/AZM versus CRO. This will be 
done using agar well diffusion assays to compare the inhibitory zone produced by the oropharyngeal 
microbiota obtained 14 days after receipt of either CRO/AZM or CRO for the treatment of Ng.  
The experiments will be performed using 2 different Ng clinical isolates that are susceptible to this 
inhibitory effect of oropharyngeal microbiota as assessed by the agar well diffusion assay.  
An aliquot of the oral rinse sample taken from day 14 will be used to inoculate two agar plates – a 
blood agar plate for the growth of Neisseria and a Streptococcus selective medium (Oxoid, 
Basingstoke, UK) for the growth of Streptococci. Three randomly selected individual colonies from each 
of these plates will be individually placed in wells in new agar plates (again Neisseria or streptococcal 
agars). These will then have one of the Ng isolates plated over them. The inhibitory effect of the 
commensals on Ng growth will be measured 24 hour later. 
We will use paired samples t-tests to compare the zone of inhibition (assessed by imageJ: 
https://imagej.nih.gov/ij/download.html) between the CRO and CRO/AZM groups. 
5.4.2. Laboratory testing at external laboratories 
Resistome and microbiome profiling will be performed outsourced to an academic or commercial 
laboratory. All specimens taken for that purpose will be stored at the CRL at -80°C.   
All laboratory procedures relating to the Ng-morbidostat will be carried out at the ITM STI Laboratory.       


23 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
5.4.3. Microbiome and resistome laboratory procedures  
• 
DNA extraction: Metagenomic DNA will be extracted from anorectal swabs via phenol-
chloroform bead-beating protocol as described previously [62]. Anorectal and oropharyngeal 
DNA will be extracted from swabs 
• 
DNA sequencing: oropharyngeal and anorectal DNA will be sequenced by whole metagenome 
shotgun sequencing. This sequencing work will be outsourced. 
 
All analyses of clinical trial samples will be carried out in compliance with Good Clinical Laboratory 
Practice (GCLP). A Lab Analytical Plan will be available, in line with WHO-GCLP. 
 
6. STUDY INVESTIGATIONAL PRODUCT 
6.1 Purchasing, preparation and administration 
The study will use two Investigational Medicinal Products (IMPs): 
1) Ceftriaxone 1g IM (intramuscularly) + lidocaine 35 mg - single dose at day 0 
2) Azithromycin 2g PO (per os) - single dose at day 0 
These medications (CRO and AZM) will be purchased from a local pharmacy and stored at room 
temperature in a locked facility at ITM in accordance with good distribution and storage practice..  
Instructions will be provided as to how to take the azithromycin PO and this will be given under 
observation [59]. The CRO will be prepared according to the product insert (SmPC) and administered 
IM by a qualified practitioner.  
The IMPs have EC-marketing approval and are available upon medical prescription in Belgian 
pharmacies. SmPC documentation is available for these products. 
Further details from the BCFI pertaining to the IMP is available from:  
- 
CRO: https://www.bcfi.be/nl/chapters/12?frag=9720  
- 
AZM: https://www.bcfi.be/nl/chapters/12?frag=9837  
 
6.2 Participant compliance monitoring 
The AZM will be given under observation. CRO will be administered by site staff. 
 
6.3 Prior and concomitant therapy 
Concomitant drugs will be noted during the visits on day 0 and day 14. Patients will be asked to contact 
the study team in case they require administration of an antibiotic throughout the study. The use of 
this antibiotic will be noted and the patient will exit the study at this point and be replaced by a new 
participant.  
 
6.4 Packaging 
The IMPs will retain their standard packaging. However a study label will be added to the commercial 
packaging with the following information: 
- 
Study name + EudraCT number 
- 
Contact details of the PI 
- 
FOR CLINICAL TRIAL USE ONLY 
- 
Sponsor name (Institute of Tropical Medicine) 


24 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
This study label will be provided by the sponsor and the relabeling procedure will be performed 
according to the study-specific relabeling SOP. We will adhere to a stringent relabeling procedure 
under the supervision of a qualified pharmacist. The information on the study labels will be available 
in German, French and Dutch.  
 
6.5 Reception, storage, dispensing and return 
The IMPs will be stored at 15-25 °C as required for these products and as described in the SmPC. This 
will be done under the supervision of a qualified pharmacist. 
 
7. SAFETY ASSESSMENT 
7.1 (Serious) Adverse Events 
(Serious) Adverse Events will be collected, monitored and recorded starting from the screening visit 
and up till the moment the patient exits the study.  
Adverse Events 
Since the IMPs are already marketed and widely used,  a predefined list of AE (as listed below) will be 
reviewed at every study visit and reported in the source documents. These events will also be recorded 
in the eCRF (electronic Case Report Form):  
- 
Diarrhea (CRO, AZM) 
- 
Skin rash (CRO) 
- 
Headache (CRO, AZM) 
- 
Dizziness (CRO) 
- 
Nausea or vomiting (CRO, AZM) 
- 
Abdominal pain (AZM) 
- 
Pruritus (CRO) 
- 
Pain at injection site (CRO) 
 
It is the Investigator’s responsibility to adequately and timely report Adverse Events to the local EC’s 
in compliance with applicable local regulations.  
 
Serious Adverse Events 
A Serious Adverse Event (SAE) is any adverse event/experience occurring at any study drug dose that 
results in any of the following outcomes: 
• 
Death; 
• 
Life threatening (participant at immediate risk of death); 
• 
Requires in-patient hospitalization or prolongation of existing hospitalization; 
• 
Results in congenital anomaly/birth defect; 
• 
Results in a persistent or significant disability or incapacity; 
• 
An important medical event that may not result in death, be life threatening, or require 
hospitalization may be considered an SAE when, based upon appropriate medical judgment, the 
event may jeopardize the participant and may require medical or surgical intervention to prevent 
one of the outcomes listed in this definition. 
 
 
 


25 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
 
 
All SAEs must be: 
• 
recorded on the appropriate SAE report form 
• 
followed through resolution by a study clinician 
• 
reviewed and evaluated by a study clinician. 
• 
Follow-up until resolution (by sending updates on the SAE form) 
 
Severity, relationship of event to study drug and outcome 
All SAE’s will be assessed by the clinician using a predefined grading system:  
1. Mild: events require minimal or no treatment and do not interfere with the participant’s daily 
activities. 
2. Moderate: events result in a low level of inconvenience or concern with the therapeutic 
measures.  Moderate events may cause some interference with functioning. 
3. Severe: events interrupt a participant’s usual daily activity and may require systemic drug 
therapy or other treatment.  Severe events are usually incapacitating. 
4. Life-threatening: Participant at risk for death at the time of the event 
 
Changes in the severity of an SAE will be documented to allow an assessment of the duration of the 
event at each level of intensity to be performed.  Adverse events characterized as intermittent require 
documentation of onset and duration of each episode. 
 
Assessment of causality   
The investigator is obliged to assess the relationship between investigational product and the 
occurrence of each SAE. The investigator will use clinical judgement to determine the relationship. 
Alternative causes, such as natural history of the underlying diseases, concomitant therapy, other risk 
factors, and the temporal relationship of the event to the IMP will be considered and investigated. The 
investigator will also consult the drug information as needed in the determination of his/her 
assessment. 
 
The relationship of an adverse event to study drug is to be assessed according to the following 
definitions and can only be done by the study physician:  
1. Definitely unrelated: Reserved for those events which cannot be even remotely related to 
study participation (e.g. injury caused by a third party). 
2. Unlikely: There is no reasonable temporal association between the study drug and the AE 
and the event could have been produced by the participant's clinical state or other modes of 
therapy administered to the participant.  
3. Possible: The suspected AE may or may not follow a reasonable temporal sequence from 
study drug administration but seems to be the type of reaction that cannot be dismissed as 
unlikely. The event could have been produced or mimicked by the participant's clinical state 
or by other modes of therapy concomitantly administered to the participant. 
4. Likely: The suspected adverse event follows a reasonable temporal sequence from study 
drug administration, abates upon discontinuation of the drug, and cannot be reasonably 
explained by the known characteristics of the participant's clinical state.  
5. Definitely related: Reserved for those events which have no uncertainty in their relationship 
to test drug administration: this means that a re-challenge was positive. 
 
 


26 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
 
The outcome of each SAE must be assessed at the same visit and according to the following 
classification: 
Recovered: The participant recovered from the event with no residual problems.  
Not yet recovered: This outcome can only be used for Serious Adverse Events.  The event no longer 
meets a ‘Serious’ criterion, but medical event is not yet completely cured. The remaining event 
should be listed as a separate adverse event in the adverse event table, and with its own outcome.  
Permanent damage: The event has resulted in permanent impairment.  
Ongoing: the participant is continued to be followed for the event. 
Death: The participant died. This term should only be used for the event which resulted in death. 
Any other events which were present at the time of death, but were not the cause of death, should 
be listed as ‘Ongoing’. 
Unknown: The participant cannot be traced and no final outcome for the event could be 
determined. 
There may be situations when an SAE has occurred, and the investigator has minimal information to 
include in the initial report to the Sponsor. However, it is very important that the investigator always 
makes an assessment of causality for every event prior to transmission of the report to the Sponsor. 
The investigator may change his/her opinion of causality in light of follow-up information, amending 
the SAE-report form accordingly. 
All SAE’s whether or not deemed drug related or expected must be reported immediately or within 24 
hours (one working day) using the SAE Notification Form by email to: 
 
 
Clinical Trials Unit 
 
Institute of Tropical Medicine 
 
Nationalestraat 155 
 
B-2000 Antwerp – Belgium 
 
Email: pharmacovigilance@itg.be 
 
Line listings of all reported SAE’s will be sent to the IRB of the ITM and the independent EC, on a yearly 
basis. An annual safety and status report will be sent to the Belgian Competent Authorities. 
 
Suspected and unexpected SAE’s (SUSARs) must also be reported by the sponsor to the Belgian 
Competent Authority (for death and life-threatening, within 7 days, for all other SUSARs within 15 
days). Reporting and management of SUSARs is described in the sponsor SOP on ‘Safety Reporting’.  
 
A study safety checklist SOP will be prepared describing all responsibilities and actions that are 
required in case of an SAE. 
 
7.2 Data and Safety Monitoring Board 
As this study is a low intervention clinical trial with investigational products that will be used according 
to their market registration, no data safety monitoring board will be installed and not independent 
safety monitor will be appointed. 
 
 
 
 


27 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
8. STATISTICAL METHODS 
The statistical analysis will be described in the statistical analysis plan (SAP), written by the 
biostatistician, which is binding and will be finalized before database lock or before any other 
analysis takes place. 
 
8.1 Study hypotheses and hypothesis 
The primary hypothesis of this study is that AZM/CRO therapy for Ng results in an increase in the 
abundance of macrolide resistance determinants compared to CRO therapy.  
 
8.2 Variables of interest 
 
Primary outcome  
During the bioinformatic analyses, each identified antibiotic-resistance determinant will be 
categorized at the class level using a read-based classification tool. The primary outcome will be the 
ratio of mean macrolide resistance determinants in the day 14 anorectal samples between the two 
treatment groups. This ratio will be calculated by dividing the mean normalized read count of 
macrolide- resistance determinants categorized at the class level in the CRO/AZM group by the 
corresponding mean quantity in the CRO group . 
Secondary outcomes 
1. the ratio of mean normalized read count of resistance determinants for each non-macrolide 
antibiotic class in the day 14 anorectal samples between the two treatment groups 
2. the ratio of mean normalized read count of resistance determinants for each macrolide and 
non-macrolide antibiotic class in the day 14 oropharyngeal samples between the two 
treatment groups 
3. The difference in proportion of oropharyngeal commensal Neisseria that are macrolide 
resistant between the two treatment groups at day 14. For each individual the proportion of 
oropharyngeal commensal Neisseria that are macrolide resistant will be calculated by dividing 
the number of macrolide-resistant colonies of commensal Neisseria grown on modified LB 
agar-azithromycin-1mg/L by the total number of commensal Neisseria colonies grown on 
modified LB agar without azithromycin.    
4. The difference in time until acquisition of phenotypic resistance to AZM by Ng in the 
morbidostat upon exposure to commensal Neisseria DNA extracts from each treatment group 
5. The difference in diameter of the inhibition zone of Ng during agar overlay experiments using 
oropharyngeal commensal Neisseria and Streptococci from day 14 samples from each 
treatment group 
Safety outcome 
The  number of participants with (severe) adverse events in each treatment group.  
 
8.3 Statistical methods 
 
8.3.1 Analysis populations 
The primary analysis and secondary efficacy analyses will be performed on all randomized participants 
with samples at baseline and day 14. All participants will be analyzed according to their randomization 
group.  
 


28 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
8.3.2 Baseline characteristics 
The number of participants screened and enrolled or excluded will be summarized according to reason 
for exclusion. Of the enrollees, the number of patients discontinued or lost to follow-up will be 
recorded by reason and time of discontinuation. These figures will be summarized in a CONSORT flow 
diagram. 
Patients in each treatment group will be described according to baseline characteristics. The 
description will be in terms of medians and interquartile ranges for continuous variables and using 
counts and percentages for categorical variables. Differences in baseline characteristics will be noted, 
but no formal statistical testing will be done.   
 
8.3.3 Primary analysis 
The primary analysis of assessing a difference between the mean macrolide resistance determinants 
in anorectal microbiome between the two groups will be done using permutation test. The p-value of 
the test will determine if there is a statistically significant difference between the two means at a 5% 
significance level. The estimated ratio of the two arms is be presented with a 95% confidence interval.  
 
8.3.4 Secondary and tertiary analysis 
The secondary objectives regarding mean comparisons will be done similarly to the primary analysis. 
Alternatively, a t-test in the log-transformed means of the two groups will be performed as a 
comparison to the permutation test. A 95% CI for the ratio of the two groups will be calculated using 
permutation testing.  
The difference of proportions of resistance between the two arms will be calculated with the 95% 
confidence interval of the proportion difference at each time point separately. The differences in 
acquisition of AZM resistance and diameter of inhibition zone will be done using permutation testing 
and the Wilcoxon rand sum test.  
The patient count with adverse events in the two groups will be compared using Fisher’s exact test.  
 
8.3.5 Subgroup analyses 
No a priori subgroup analyses are defined. 
 
8.3.6 Multiplicity and Missing Data 
No adjustment for multiplicity are needed. No interim analyses are planned. We estimate that 5% of 
the day 14 visits will be missed. These individuals will be dropped from the analyses, but the amount 
of missing data in each characteristic will be described.    
 
8.4 Sample size and power 
We assume that CRO/AZM will result in a 2.5 fold increase in the ratio of macrolide resistance 
determinants compared to the CRO group. This estimate is likely a conservative estimate based on a 
previous study that found a seven-fold increase in this ratio following repeated AZM mass 
administration in Niger – the MORDOR study [39]. We have used a much lower estimate as there are 
important differences between the study populations in MORDOR and this study. These include 
children in MORDOR versus adults in ResistAZM and a low vs. very high background macrolide 
consumption in MORDOR and ResistAZM, respectively [29]. 
The sample size was calculated by simulation using permutation testing with the assumed effect size 
and a 5% significance level. The minimum sample size in order to obtain at least 80% power is 20 


29 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
participant per group. Correcting for a drop-out rate of 5%, we will need 21 men in each arm to detect 
this effect size with 80% power using a two-sided significance level of α=0.05.    
 
 
9. MONITORING AND QUALITY ASSURANCE 
This study will be monitored by the ITM CTU (Clinical Trial Unit) in accordance with regulations 
applicable to clinical trials, including ICH-GCP (Good Clinical Practice) and WHO-GCLP, and sponsor-
specific SOPs. The PI and involved site research staff will allocate adequate time and resources for 
such monitoring activities. The investigator will also ensure that the monitor or QA reviewer is given 
access to all the above noted study-related documents and study-related facilities (e.g. pharmacy, 
diagnostic laboratory, etc.) and has adequate space and resources to conduct monitoring and source 
data verification (SDV). 
A monitoring plan will be written to describe monitoring responsibilities and activities in detail 
(including percentage of SDV, timing and frequency of site visits, follow-up of findings and protocol 
deviations).  
The sponsor will inform the Investigators concerned immediately upon notification of a pending study 
centers inspection by any regulatory authority or funder. Likewise, the investigator will inform the 
sponsor of any pending inspection. 
Laboratory quality control and quality assurance:                                                                                                             
ITM will ensure that all laboratory activities including specimen transport, processing, testing, result 
reporting and storage will be conducted in accordance with the clinical trial quality requirements. The 
laboratory will perform testing according to the SOPs which are documented in the laboratory 
analytical plan and will be conducted in compliance with Good Clinical Laboratory Practice Standards 
(GCLP), and EN-ISO 15189. Reports of laboratory test results will be forwarded to the study physician 
as soon as the result is available. 
Trial management Group: 
The PI, STI laboratory manager, study doctors and a CTU-representative will constitute the trial 
management group.  Additional people (statistician, data manager, data reviewer, QA officer…) might 
join the meeting if necessary. 
 
10. DATA MANAGEMENT 
Data Management will be done in compliance with GDPR, GCP guidelines and FDA 21 CFR part 11 
regulations and performed by the data manager in collaboration with the study staff at the site.  
A Data Management Plan will be prepared, with following essential data management aspects: 
Types of data collected 
 
 
 
 
 
 
 
                   
Individual, participant-level clinical trial and behavioral data will be collected by the clinical site team 
and held in patient files at the site. Only data defined by the study protocol will be collected. Collected 
data will include variables from Baseline visit (de-identified trial participant code, eligibility screening, 
informed consent confirmation variables, medical history, concomitant medication, randomization, 
lab test results,  adverse events  and behavioral data), Follow-Up visit data (lab test results, 
concomitant medication, (S)AE data), behavioral data and outcome data (final outcome).  
Data collection and handling                                       
 
 
 
 
 
                  
Data will be entered at the site via study computers equipped with electronic Case Report Forms 
(eCRFs) developed with a GCP- and regulatory compliant clinical trials management software. Edit 


30 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
checks and branching logic, programmed onto the electronic forms will validate at the point of data 
entry and support quality data. The electronic forms will be tested and validated before the actual data 
collection. The study team will be trained on the use of the eCRFs, data collection and query handling 
before the study start. 
Data storage & preservation 
All the relevant study data will be retained for a minimum of twenty years and according to applicable 
regulations. The trial database will be stored on a secured server at the ITM, which is physically only 
accessible by badge by authorized IT collaborators. The ITM has procedures in place to ensure daily 
backup of the server and study computers and for long-term, secure curation and preservation of data.  
 
Confidentiality & security  
The computerized system that will be used includes a robust security architecture including encryption, 
firewalls, antivirus software and controlled user access (username, personal password and user roles 
with different privileges), tracking of edits (electronic audit trail) and the use of a trustworthy  
electronic signature. A list of authorized users of the eCRFs, with their user role will be kept at the CTU 
and updated regularly. 
Information of trial participants will be handled confidentially. A trial participant code (pseudonym) 
will be assigned to each trial participant at the earliest opportunity. Any information that could lead to 
the direct identification of the participant will not be included on the eCRFs, nor on any other paper 
documents or electronic files used for data management. The name and contact data for each 
participant will be kept separately and limited to authorized staff at the site.  
Only pseudonymized data will be distributed amongst the study team during the study. 
Both the sponsor and site will also see to it that the necessary measures are taken to ensure that all 
data management documents (such as the Data Management Plan, and procedures) and IT devices 
and equipment (computers, server) are kept secure (in closed cupboards, closed and/or badge-
controlled offices or rooms). 
 
Data integrity and quality 
Ensuring data integrity and data quality is an essential task throughout the trial, carried out by 
competent study staff who by preference are assigned to specific roles in data management and ICT 
(such as the data manager, data reviewer, monitor, IT helpdesk). The study specific Data Management 
Plan (DMP) and ICT and Data Management Standard Operating Procedures (SOPs) are amongst the 
essential documentation which will guide and support the trial conduct. Good practices during the 
design of the eCRFs, such as use of coding, check boxes, question input masks and drop-down menus 
will enhance the data quality. Aside from automatic edit checks, a specific Data Validation Plan with 
manual checks will also be performed to identify out of range data, missing data and inconsistent data. 
Medical event terms will be coded and standardized using the Medical Dictionary for Regulatory 
Activities (MedDRA).  
 
Data sharing 
After study completion and publication of results, anonymized individual participant data may be 
shared by means of a managed access procedure and along a transparent decision-making process, 
with: (i) Completion of a data request form, (ii) Evaluation by a data access committee, (iii) Data sharing 
Agreement, (iv) Secure transfer of anonymized data, and (v) Additional metadata which clarify the 
research data. Metadata will include the study protocol, questionnaire(s), data dictionary. Specifics 
about data sharing will be included in the Informed Consent Form. To this end, the ITM Data Sharing 
Policy will be adhered to.  
All research data processing will be performed in compliance with the European General Data 
Protection Regulation 2016/679 (GDPR). ITM is the controller of the data processing and the lawful 
basis for the processing of participant personal data will be the public interest. 


31 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
11. ETHICAL ISSUES 
The study design randomizes individuals to receive one of two treatment schemas – CRO and 
CRO/AZM. Treatment with CRO is the preferred therapy for Ng according to the UK, USA and 
Netherlands Ng guidelines whereas treatment with CRO/AZM is the preferred treatment according 
the Belgian and IUSTI Europe guidelines. Both treatments have been given to a large number of 
individuals without any large concerns raised about their safety or efficacy. As such we do not think 
that there is evidence to regard either or both of these treatment regimens as unethical.  
Participants will be required to return for a post-treatment visit. This is standard practice and explicitly 
recommended in the IUSTI Europe Ng treatment guidelines.  
Participants will not be renumerated, but participants will not be charged any costs additional to those 
that result from their routine case management. In particular, participants will not be charged any 
costs additional to those that result from their routine case management. In fact by virtue of not 
paying for the antimicrobials used, participating in the study will cost them less than routine care. 
 
11.1 Ethical and regulatory review 
This clinical trial will be submitted for formal review and approval to the Institutional Review Board of 
the ITM, the designated Belgian EC and Competent Authorities of Belgium (FAMPH). The trial will be 
submitted to the FAMPH pilot project for the EU Clinical Trial Regulation 536/2014, which includes 
review by an independent Ethical Committee. No study-specific interventions will take place before 
written approval by the Ethics Committee(s) has been obtained and the local regulatory requirements 
have been complied with, and the signature of the clinical study protocol of each contractual party 
involved have been obtained. 
The study will be carried out according to the principles stated in the Declaration of Helsinki as 
amended in 2013 and any further updates, all applicable national and international regulations and 
according to the most recent ICH-GCP guidelines. 
The study will also be included in the EudraCT public registry prior to the start of participant 
recruitment. 
 
11.2 Protocol amendments 
Once the final clinical study protocol has been issued and signed by the authorized signatories, it 
cannot be informally altered. Protocol amendments have the same legal status and must pass through 
the appropriate steps before being implemented. Any substantial change must be approved by all the 
bodies and EC’s that have approved the initial protocol, prior to being implemented, unless it is due 
to participant’s safety concerns (in which case the immediate implementation can be necessary for 
the sake of participant’s protection. In case modifications to the protocol or amendment are 
requested by any local EC/CA during the review process, these must be discussed and agreed upon 
with the Sponsor prior to any resubmission incorporating those changes. 
 
11.3 Informed consent 
No participant may be enrolled into the study until the Investigator or designee has obtained the 
written informed consent form (see section 5.2). 
 
 


32 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
11.4 Confidentiality 
All the participant data will be pseudonymised in all collection tools, the CRF and database by means 
of a unique subject assigned participant study number. The study documents which can identify the 
participants, e.g. the ICF’s, , study source medical record, will only be accessible to the relevant study 
staff, study monitors, auditors and inspectors under confidentiality agreements.  
Privacy by Design principles will be adhered to with regard to personal data processing and maximizing 
confidentiality. Data collection will be minimized as much as possible and will be proportionate to 
achieve the goals of the study. Each participant will be assigned a unique personal identification 
number (PID), which will be used on all study forms. This identification number will be linked to the 
person’s name and contact information in a central “participant identification list”. The identification 
list and documents containing the names and/or signatures of participants (such as consent forms) will 
be kept separately from all other study documents containing participant data, that will only be 
identified by the participant identification numbers. All study documents will be stored in lockable 
rooms or cabinets with access limited to study staff. 
Names of the participants will not appear on any reports or publications resulting from this study. The 
findings from this study may be published in a medical/scientific journal. The study participants will 
never be identified by name. After the study is completed, participants will be informed of the study 
results by the Principal Investigator or designee. 
The Sponsor provides all study documents to the Investigators and his/her appointed staff in 
confidence. Materials may not be disclosed to any party not involved in the study, unless written 
permission from the Sponsor or ITMs Data Access Committee with regard to the sharing of data for 
secondary research. 
 
11.5 Risks and benefits 
No major risks are anticipated for the participants. There may be a small risk of breach of privacy during 
the study. Because we are collecting data pertaining to sexual behavior and STIs, any breach of privacy 
could have serious consequences for participants. We are aware of this risk and have designed the 
study in a way to minimize this risk. In particular we will follow strict protocols to maintain patient 
confidentiality (details provided in section 11.4). 
The principal benefit for the participants is to contribute to optimizing the treatment of Ng in a way 
which minimizes the emergence and spread of AMR in Ng and other bacteria. 
 
 
11.6 Compensation for participation 
There will be no direct compensation for the study participation. 
 
11.7 Insurance 
As required by the Belgian law on experiments on the human person of May 7th 2004, the sponsor has 
obtained a no-fault liability insurance (with Amlin Insurance S.E., insurance policy number LXX099279) 
covering any harm, injury or (material) damage which may occur to study participants and which may 
be directly or indirectly caused by their participation in the trial. The insurance provisions will also be 
mentioned during the informed consent discussion and in the ICF. 
 


33 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
12. DISSEMINATION OF RESULTS, INTELLECTUAL PROPERTY 
All study documents are provided by the Sponsor to the Investigators and his/her appointed staff in 
confidence. None of this material may be disclosed to any party not directly involved with the study, 
without written permission from the Sponsor.  
Reporting and publication of the study data will be done in accordance with the CONSORT statement 
(check http://www.consort-statement.org/consort-statement/) and ITM’s publication policy. 
 
13. ARCHIVING 
The sponsor and Investigator must maintain adequate and accurate records to enable the conduct of 
the study to be fully documented and the study data to be verified. The relevant (essential) documents 
are those documents which individually and collectively permit to assess the conduct of the trial, the 
quality of the data produced and the compliance with GCP standards and applicable regulatory 
requirements. The Investigator’s File should at least contain all the (essential) documents as listed in 
the procedure “Set up and maintenance of the Investigator Trial File”. A copy of all source data and 
Case Report Forms must always be kept on site. The PI is responsible for ensuring a secure and 
appropriate location for his Investigator’s File and any other trial related documentation present at 
site, as well as for ensuring that only site staff that is competent and delegated to work for the trial 
has got access to the files. 
All the relevant study documentation should be retained for a minimum of 20 years after completion 
of the study, as set out by the current Belgian law. The Sponsor should be informed prior to 
destruction of the files. 
After completion of the study, the IF will remain available for internal audits and/or inspections of 
regulatory authorities for a period of 25 years. 
 
14. REFERENCES 
 
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Blomquist PB, Miari VF, Biddulph JP, Charalambous BM. Is gonorrhea becoming 
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ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
10.1128/AAC.00149-15. PubMed PMID: 26787702; PubMed Central PMCID: 
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7. 
Zhao S, Duncan M, Tomberg J, Davies C, Unemo M, Nicholas RA. Genetics of 
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Wadsworth CB, Arnold BJ, Sater MRA, Grad YH. Azithromycin Resistance through 
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Fiore MA, Raisman JC, Wong NRH, Hudson AO, Wadsworth CB. Exploration of the 
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Unemo M, Workowski K. Dual antimicrobial therapy for gonorrhoea: what is the role 
of azithromycin? The Lancet Infectious Diseases. 2018;18(5):486-8. 
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Barbee LA, Soge OO, Holmes KK, Golden MR. In vitro synergy testing of novel 
antimicrobial combination therapies against Neisseria gonorrhoeae. J Antimicrob Chemother. 
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17. 
Pereira R, Cole MJ, Ison CA. Combination therapy for gonorrhoea: in vitro synergy 
testing. J Antimicrob Chemother. 2013;68(3):640-3. Epub 2012/11/16. doi: 
10.1093/jac/dks449. PubMed PMID: 23152483. 
18. 
Unemo M, Bradshaw CS, Hocking JS, de Vries HJC, Francis SC, Mabey D, et al. 
Sexually transmitted infections: challenges ahead. Lancet Infect Dis. 2017;17(8):e235-e79. 
Epub 2017/07/14. doi: 10.1016/S1473-3099(17)30310-9. PubMed PMID: 28701272. 
19. 
Cyr SS, Barbee L, Workowski KA, Bachmann LH, Pham C, Schlanger K, et al. 
Update to CDC's Treatment Guidelines for Gonococcal Infection, 2020. Morbidity and 
Mortality Weekly Report. 2020;69(50):1911. 
20. 
Nederlandse Vereniging voor Dermatologie en Venereologie (NVDV) en de 
Nederlandse Vereniging voor Medische Microbiologie (NVMM). Seksueel Overdraagbare 
Aandoeningen Multidisciplinaire Richtlijn (2017 Update). 2017. 


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21. 
Kenyon C, De Baetselier I, Wouters K. Screening for STIs in PrEP cohorts results in 
high levels of antimicrobial consumption. Int J STDs & AIDS. 2020. 
22. 
Cole M, Quaye N, Chisholm S, Ison C, Laar M, Spiteri G. Gonococcal antimicrobial 
susceptibility surveillance in Europe 2010: European Centre for Disease Prevention and 
Control; 2015. 
23. 
Gernert KM, Seby S, Schmerer MW, Thomas IV JC, Pham CD, St Cyr S, et al. 
Azithromycin susceptibility of Neisseria gonorrhoeae in the USA in 2017: a genomic analysis 
of surveillance data. The Lancet Microbe. 2020;1(4):e154-e64. 
24. 
Rambaran S, Naidoo K, Dookie N, Moodley P, Sturm AW. Resistance Profile of 
Neisseria gonorrhoeae in KwaZulu-Natal, South Africa Questioning the Effect of the 
Currently Advocated Dual Therapy. Sex Transm Dis. 2019;46(4):266-70. Epub 2019/01/19. 
doi: 10.1097/OLQ.0000000000000961. PubMed PMID: 30657464. 
25. 
Buono SA, Watson TD, Borenstein LA, Klausner JD, Pandori MW, Godwin HA. 
Stemming the tide of drug-resistant Neisseria gonorrhoeae: the need for an individualized 
approach to treatment. J Antimicrob Chemother. 2015;70(2):374-81. doi: 10.1093/jac/dku396. 
PubMed PMID: 25331059. 
26. 
Fifer H, Natarajan U, Jones L, Alexander S, Hughes G, Golparian D, et al. Failure of 
dual antimicrobial therapy in treatment of gonorrhea. New England Journal of Medicine. 
2016;374(25):2504-6. 
27. 
Hsia Y, Lee BR, Versporten A, Yang Y, Bielicki J, Jackson C, et al. Use of the WHO 
Access, Watch, and Reserve classification to define patterns of hospital antibiotic use 
(AWaRe): an analysis of paediatric survey data from 56 countries. Lancet Glob Health. 
2019;7(7):e861-e71. Epub 2019/06/16. doi: 10.1016/S2214-109X(19)30071-3. PubMed 
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28. 
Organization WH. The 2019 WHO AWaRe classification of antibiotics for evaluation 
and monitoring of use. World Health Organization, 2019. 
29. 
Kenyon C, Van Dijck C, Basil S. Is there a resistance-threshold for macrolide 
consumption? Positive evidence from an ecological analysis of resistance data from 
Streptococcus pneumoniae, Treponema pallidum and Mycoplasma genitalium. medRxiv. 
2020. doi: https://doi.org/10.1101/2020.10.13.20212043. 
30. 
Kenyon C. Prevalence of macrolide resistance in Treponema pallidum is associated 
with macrolide consumption. J Med Microbiol. 2019;68(2):119-23. Epub 2018/12/07. doi: 
10.1099/jmm.0.000885. PubMed PMID: 30520715. 
31. 
Kenyon C. We need to consider collateral damage to resistomes when we decide how 
frequently to screen for chlamydia/gonorrhoea in PrEP cohorts. AIDS. 2019;33:155-7. 
32. 
Laumen JGE, Van Dijck C, Abdellati S, Manoharan-Basil SS, De Baetselier I, 
Martiny D, et al. Markedly reduced azithromycin and ceftriaxone susceptibility in commensal 
Neisseria species in clinical samples from Belgian men who have sex with men. Clin Infect 
Dis. 2020. Epub 2020/05/14. doi: 10.1093/cid/ciaa565. PubMed PMID: 32402070. 
33. 
Jernberg C, Lofmark S, Edlund C, Jansson JK. Long-term ecological impacts of 
antibiotic administration on the human intestinal microbiota. ISME J. 2007;1(1):56-66. doi: 
10.1038/ismej.2007.3. PubMed PMID: 18043614. 
34. 
Jakobsson HE, Jernberg C, Andersson AF, Sjolund-Karlsson M, Jansson JK, 
Engstrand L. Short-term antibiotic treatment has differing long-term impacts on the human 
throat and gut microbiome. PLoS One. 2010;5(3):e9836. doi: 10.1371/journal.pone.0009836. 
PubMed PMID: 20352091; PubMed Central PMCID: PMC2844414. 
35. 
Korpela K, Salonen A, Virta LJ, Kekkonen RA, Forslund K, Bork P, et al. Intestinal 
microbiome is related to lifetime antibiotic use in Finnish pre-school children. Nat Commun. 
2016;7:10410. doi: 10.1038/ncomms10410. PubMed PMID: 26811868; PubMed Central 
PMCID: PMCPMC4737757. 


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36. 
Sjolund M, Wreiber K, Andersson DI, Blaser MJ, Engstrand L. Long-term persistence 
of resistant Enterococcus species after antibiotics to eradicate Helicobacter pylori. Ann Intern 
Med. 2003;139(6):483-7. PubMed PMID: 13679325. 
37. 
Malhotra-Kumar S, Lammens C, Coenen S, Van Herck K, Goossens H. Effect of 
azithromycin and clarithromycin therapy on pharyngeal carriage of macrolide-resistant 
streptococci in healthy volunteers: a randomised, double-blind, placebo-controlled study. The 
Lancet. 2007;369(9560):482-90. 
38. 
Sjolund M, Tano E, Blaser MJ, Andersson DI, Engstrand L. Persistence of resistant 
Staphylococcus epidermidis after single course of clarithromycin. Emerg Infect Dis. 
2005;11(9):1389-93. doi: 10.3201/eid1109.050124. PubMed PMID: 16229767; PubMed 
Central PMCID: PMCPMC3310621. 
39. 
Doan T, Worden L, Hinterwirth A, Arzika AM, Maliki R, Abdou A, et al. Macrolide 
and Nonmacrolide Resistance with Mass Azithromycin Distribution. N Engl J Med. 
2020;383(20):1941-50. Epub 2020/11/12. doi: 10.1056/NEJMoa2002606. PubMed PMID: 
33176084; PubMed Central PMCID: PMCPMC7492079. 
40. 
Doan T, Arzika AM, Hinterwirth A, Maliki R, Zhong L, Cummings S, et al. Macrolide 
Resistance in MORDOR I - A Cluster-Randomized Trial in Niger. N Engl J Med. 
2019;380(23):2271-3. Epub 2019/06/06. doi: 10.1056/NEJMc1901535. PubMed PMID: 
31167060; PubMed Central PMCID: PMCPMC6518950. 
41. 
Chisholm SA, Mouton JW, Lewis DA, Nichols T, Ison CA, Livermore DM. 
Cephalosporin MIC creep among gonococci: time for a pharmacodynamic rethink? J 
Antimicrob Chemother. 2010;65(10):2141-8. Epub 2010/08/10. doi: 10.1093/jac/dkq289. 
PubMed PMID: 20693173. 
42. 
Connolly KL, Eakin AE, Gomez C, Osborn BL, Unemo M, Jerse AE. 
Pharmacokinetic Data Are Predictive of In Vivo Efficacy for Cefixime and Ceftriaxone 
against Susceptible and Resistant Neisseria gonorrhoeae Strains in the Gonorrhea Mouse 
Model. Antimicrob Agents Chemother. 2019;63(3). Epub 2019/01/16. doi: 
10.1128/AAC.01644-18. PubMed PMID: 30642924; PubMed Central PMCID: 
PMCPMC6395893. 
43. 
Benoun JM, Labuda JC, McSorley SJ. Collateral Damage: Detrimental Effect of 
Antibiotics on the Development of Protective Immune Memory. MBio. 2016;7(6). Epub 
2016/12/22. doi: 10.1128/mBio.01520-16. PubMed PMID: 27999159; PubMed Central 
PMCID: PMCPMC5181774. 
44. 
Lipsitch M, Samore MH. Antimicrobial use and antimicrobial resistance: a population 
perspective. Emerg Infect Dis. 2002;8(4):347-54. Epub 2002/04/25. doi: 
10.3201/eid0804.010312. PubMed PMID: 11971765; PubMed Central PMCID: 
PMCPMC2730242. 
45. 
Kong FYS, Hatzis CL, Lau A, Williamson DA, Chow EPF, Fairley CK, et al. 
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analysis of randomized controlled trials. J Antimicrob Chemother. 2020;75(11):3109-19. 
Epub 2020/08/05. doi: 10.1093/jac/dkaa300. PubMed PMID: 32747940. 
46. 
Van Dijck C, Tsoumanis A, Rotsaert A, Vuylsteke B, Van den Bossche D, Paeleman 
E, et al. Does an antibacterial mouthwash prevent sexually transmitted infections in men who 
have sex with men taking HIV pre-exposure prophylaxis? A randomised, placebo controlled, 
crossover trial. . Lancet Infec Dis. 2021. 
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Laumen JGE, Van Dijck C, Abdellati S, Manoharan-Basil SS, De Baetselier I, 
Martiny D, et al. Markedly Reduced Azithromycin and Ceftriaxone Susceptibility in 
Commensal Neisseria Species in Clinical Samples From Belgian Men Who Have Sex With 
Men. Clin Infect Dis. 2021;72(2):363-4. Epub 2021/01/28. doi: 10.1093/cid/ciaa565. PubMed 
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ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
48. 
de Block T, Laumen JGE, Van Dijck C, Abdellati S, De Baetselier I, Manoharan-Basil 
SS, et al. WGS of commensal Neisseria reveals acquisition of a new ribosomal protection 
protein (MsrD) as a possible explanation for high level azithromycin resistance in Belgium. 
Pathogens. 2021;10(3):384. 
49. 
Caselli E, Fabbri C, D'Accolti M, Soffritti I, Bassi C, Mazzacane S, et al. Defining the 
oral microbiome by whole-genome sequencing and resistome analysis: the complexity of the 
healthy picture. BMC Microbiol. 2020;20(1):120. Epub 2020/05/20. doi: 10.1186/s12866-
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50. 
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Construction and optimization of a ‘NG Morbidostat’-An automated continuous-culture 
device for studying the pathways towards antibiotic resistance in Neisseria gonorrhoeae. 
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58. 
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So M, Rendon MA. Tribal warfare: Commensal Neisseria kill pathogen Neisseria 
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62. 
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ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
2013;8(11):e78822. doi: 10.1371/journal.pone.0078822. PubMed PMID: 24236055; PubMed 
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15. LIST OF ABBREVIATIONS 
 
AMR 
Antimicrobial resistance 
AZM  
Azithromycin 
CA 
Competent Authorities 
CASI  
Computer Assisted Self-Interview 
CI 
Coordinating Investigator 
CRL 
Clinical Reference Laboratory, ITM 
CRO 
Ceftriaxone 
Ct 
Chlamydia trachomatis 
DMP 
Data Management Plan 
DVP 
Data Validation PlanD 
(e-)CRF 
(electronic) Case Report Form 
EC 
Ethical Committee 
ECG 
Electrocardiogram 
EUCAST 
European Committee on Antimicrobial Susceptibility Testing 
FAMHP 
Federal Agency on Medicines and Health Products  
GCP 
Good Clinical Practice 
GCLP 
Good Clinical Laboratory Practice 
GDPR 
General Data Protection Regulation 
HGT 
Horizontal Gene Transfer 
IM 
Intramuscularly 
IMI 
Intramuscular Injection 
ITM 
Institute of Tropical Medicine 
IC(F) 
Informed Consent (Form) 
ICH 
International Conference on Harmonization 
(I)EC 
(Independent) Ethics Committee 
IMP 
Investigational Medicinal Product 
IRB 
Institutional Review Board 
ITM 
Institute of Tropical Medicine 
LCM 
Listerine Cool Mint 
MIC 
Minimum Inhibitory Concentration 
MTB 
Mycobacterium tuberculosis 
MSM 
Men who have Sex with Men 
NAAT 
Nucleic Acid Amplification Test 
Ng  
Neisseria gonorrhoeae 
PI 
Principal Investigator 
PrEP 
HIV Pre-Exposure Prophylaxis  


39 
ResistAZM, Protocol version 1.1, dated 3Sept2021  
 
PO  
Per Os 
QA 
Quality Assurance 
QC 
Quality Control 
RCT 
Randomized Clinical Trial 
(S)AE 
(Serious) Adverse Event 
SAP 
Statistical Analysis Plan 
SDV 
Source Data Verification 
STI 
Sexually transmitted infection 
SOP 
Standard Operating Procedure 
TMG 
Trial Management Group 
WHO 
World Health Organization 
 
 

SAP_001.pdf: 
 
 
1 
 
ResistAZM - Statistical Analysis Plan 
 
09-May-2022 
 
Statistical Analysis Plan 
 
An open-label randomized controlled trial 
comparing the effect of ceftriaxone plus 
azithromycin versus ceftriaxone for the 
treatment of Neisseria gonorrhoeae on the 
resistome  
 
ResistAZM trial 
 
Date:  09 May 2022 
 
Author:  
Achilleas Tsoumanis 
 
 
Biostatistician, Institute of Tropical Medicine, Antwerp, Belgium 
 
Approved by:  
Coordinating Investigator 
 
 
 
Chris Kenyon 
 
 
 
 
 
 
 
 
 
Date: ____ / _____ / _____ 
 
 
 
 
 
 
______________________________ 
 
 
 
 
 
 
Table of Contents 
 
1. 
Introduction ............................................................................................................ 3 
2. 
Study design and objectives ................................................................................... 3 
2.1. 
Study design .................................................................................................... 3 
2.2. 
Study objectives and endpoints ....................................................................... 3 
3. 
Definitions and analysis strategy ........................................................................... 4 
4. 
Description of the study population ....................................................................... 5 
4.1. 
Patient accounting ........................................................................................... 5 
4.2. 
Description of the study population ................................................................ 5 
5. 
Description of patient populations and outcomes .................................................. 6 
5.1. 
Patient populations .......................................................................................... 6 
5.1.1. 
Intention to treat (ITT) analysis ............................................................... 6 
5.1.2. 
Per protocol (PP) analysis ........................................................................ 6 
5.2. 
Outcomes ......................................................................................................... 7 
6. 
Statistical Methods ................................................................................................. 8 
6.1. 
Primary Efficacy Objective ............................................................................. 8 


 
 
2 
 
ResistAZM - Statistical Analysis Plan 
 
09-May-2022 
6.2. 
Secondary Efficacy Objectives ....................................................................... 8 
6.3. 
Exploratory Efficacy Objectives ..................................................................... 8 
6.4. 
Safety Objectives............................................................................................. 8 
6.5. 
Other aspects ................................................................................................... 8 
a. 
Subgroup analyses ................................................................................... 8 
b. 
Multiplicity .............................................................................................. 8 
c. 
Interim analyses ....................................................................................... 8 
d. 
Missing data ............................................................................................. 8 
 
 
 
 


 
 
3 
 
ResistAZM - Statistical Analysis Plan 
 
09-May-2022 
1. Introduction 
 
This Statistical Analysis Plan (SAP) provides a detailed and comprehensive description 
of the main, pre-planned analyses for the study. "An open label randomized controlled 
trial comparing the effect of ceftriaxone plus azithromycin versus ceftriaxone for the 
treatment of Neisseria gonorrhoeae (Ng) on the resistome". The purpose of this study 
is to examine whether dual therapy with ceftriaxone (CRO)/azithromycin (AZM) 
compared to monotherapy with CRO increases the probability of macrolide resistance 
emerging in Ng and other bacteria. The study conduct is described in the Protocol 
(EudraCT 2021-003616-10). 
 
These planned analyses will be performed by the statistician(s) at the Clinical Trials 
Unit of the Institute of Tropical Medicine (Antwerp) in collaboration with the research 
consortium. The analysis results will be described in a statistical analysis report to be 
used as the basis of the primary research publications. This document describes 
statistical methods for the primary and secondary objectives of the study as defined by 
the protocol. Additional analyses may be performed and are not covered in the current 
analysis plan.  
 
This analysis plan will be finalized and approved before database lock. Major changes 
in the statistical methodology used for the main and pre-planned analyses from this 
SAP will require detailed description and justification in the statistical analysis report. 
The final analysis datasets, programs, and outputs are archived following good clinical 
practice guidelines (ICH E9). 
 
2. Study design and objectives 
 
2.1. Study design 
 
This is an open-label, single-center, randomized controlled trial in men who have sex 
with men (MSM) attending the ITM HIV/STI clinic. A total of 42 subjects will be 
recruited and randomized (1:1) to either a dual-therapy regime CRO/AZM (n = 21) or 
to a monotherapy regime CRO (n = 21). MSM attending the HIV/STI Outpatient Clinic 
of the ITM with a diagnosis of symptomatic or asymptomatic urethritis, proctitis, or 
pharyngitis Ng will be invited for a screening visit by a member of the site research 
team. During the screening visit, inclusion and exclusion criteria will be evaluated. All 
MSM with a lab-confirmed diagnosis (NAAT or positive by Gram/methylene blue 
stain) who have signed the informed consent will be randomized via a computer-
generated schema to either dual-therapy (CRO/AZM) or monotherapy (AZM). In both 
arms, participants will be followed up in an identical fashion and will be asked to give 
the same number of samples.  
 
2.2. Study objectives and endpoints 
The primary hypothesis of this study is that AZM/CRO therapy for Ng, results in an 
increase in the abundance of macrolide resistance determinants compared to CRO 
therapy.  
 
Primary objective:  


 
 
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Assess if there is a difference in the mean macrolide resistance determinants in the 
anorectal microbiome of the CRO/AZM group compared to those in the CRO group at 
14 days post-treatment.   
 
 
Secondary objectives:  
 
1) Assess if there is a difference in the mean non-macrolide resistance determinants 
in the anorectal microbiome of the CRO/AZM group compared to those in the CRO 
group at 14 days post-treatment.  
 
2) Assess if there is a difference in the mean macrolide and non-macrolide resistance 
determinants in the oropharyngeal microbiome of the CRO/AZM group to those 
in the CRO group at 14 days post-treatment. 
  
3) Assess if there is a difference in the proportion of oropharyngeal Neisseria spp. 
that are macrolide resistant in the CRO/AZM group compared to the CRO group at 
day 0 and day 14 and the CRO/AZM group at day 0.  
 
4) Assess if the DNA extracts from the oropharyngeal Neisseria cultures obtained 
from the CRO/AZM group on day 14 result in a more rapid acquisition in AZM 
resistance than those from the CRO group.  
 
5)  Assess if the oropharyngeal commensals Neisseria (Gram-negative) and  
Streptococci (Gram-positive) obtained from the CRO/AZM group at day 14 have 
less of an inhibitory effect on the growth of Ng than those obtained from the CRO 
group.  
 
Secondary objective 2 will not be analyzed because it was decided not to perform 
oropharyngeal microbiome analyses among study participants. Secondary objectives 3-
5 will also not be a part of the main study analysis and will be part of a lab sub-study.  
 
Exploratory objective 
Assess if there is a difference in the mean macrolide and non-macrolide resistance 
determinants in the anorectal microbiome of the CRO/AZM group compared to those 
in the CRO groups at 14 days post-treatment, adjusting for the respective values at day 
0. Additional microbiome analyses, including alpha and beta diversity, will be 
performed by the bioinformatician and not by the study statistician.  
 
 
 
3. Definitions and analysis strategy 
 
Resistance determinants: The number of genes conferring resistance to specific 
antibiotic classes will be calculated during the microbiome analysis and the results will 
be provided to the study statistician as a separate database. The database will contain 
unique information such as subject id, visit in order to be able to combine with the main 
study database.  
 


 
 
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Macrolide resistance determinants: The number of genes that confer resistance to any 
of the antibiotics of the macrolide-lincosamide-streptogramin (MLS) category 
 
Non-macrolide resistance determinants: The number of genes conferring resistance to 
different classes of antibiotics (one column per category). The categories included are:  
• Aminocoumarins 
• Aminoglycosides 
• Bacitracin 
• Betalactams 
• Cationic antimicrobial peptides 
• Elfamycins 
• Fluoroquinolones 
• Metronidazole 
• Rifampin 
• Sulfonamides 
• Tetracyclines 
• Trimethoprim 
Resistance in any combination of the above groups will be coded as multi-drug 
resistance 
 
 
Total number of reads: The total number of sequencing reads per sample. This 
information will be used as a denominator and as an offset in the analyses. A value total 
reads of 0 would indicate an incorrectly taken swab or a lab failure to amplify the 
collected DNA. Samples with insufficient sequencing depth will be excluded from the 
analysis. The threshold sequencing depth will be set based on the sequencing data from 
all day 14 anorectal samples, before unblinding. 
 
Normalized resistance determinants: The number of (non-)macrolide resistance reads 
will be divided by the total number of bacterial reads to calculate the normalized 
resistance determinants. This quantity could be multiplied by 106 to present results in 
normalized determinants per million reads.  
 
 
4. Description of the study population 
 
4.1. Patient accounting 
Details of participants who are randomized, those who withdraw from the study after 
randomization and those who are lost to follow-up will be summarized in a CONSORT 
flow diagram, together with the reason of exclusion or discontinuation. The number 
(%) of participants attending scheduled follow-up visits will be reported.  
 
4.2. Description of the study population 
Participants in each treatment group will be described with respect to baseline 
characteristics. Data collected during the study visits, such as STI cases, number of sex 
partners, mouthwash use, smoking etc, will also be described. The description will be 
in terms of medians and quartiles for continuous characteristics and using counts and 


 
 
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percentages for categorical characteristics. The clinical importance of any imbalance 
will be noted, though statistical tests of significance will not be undertaken. 
 
5. Description of patient populations and outcomes 
 
5.1. Patient populations 
 
We will analyse the primary outcome both using Intention-to-Treat and Per-Protocol 
approaches, with the Intention-to-Treat approach being the primary one. In the per-
protocol analysis, only participants who receive intervention as planned and follow the 
protocol as planned are included. For the safety analysis, all patients are included in the 
intervention group they actually received (all-patients-treated approach).  
 
5.1.1. Intention to treat (ITT) analysis 
 
In the Intention-to-Treat analysis, all randomized participants who gave at least a 
sample on day 14 will be analysed according to their randomized allocation, even in 
case they receive another intervention, show protocol violations prior to or during the 
study, or are lost to follow-up.  
 
5.1.2. Per protocol (PP) analysis 
 
In the per-protocol analysis, only participants who receive intervention as planned and 
follow the protocol as planned are included.   
In Table 1 the protocol violations are classified as minor and major where minor 
violations can be included in the PP analysis population and major violations are 
excluded. 
  
 
Table 1: The protocol violations classified as minor or major violation 
 
Protocol Violation 
Major/Minor 
Violation 
Inclusion criteria 
 
1. Able and willing to provide informed consent 
Major 
2. Male sex at birth 
Major 
3. At least 18 years old 
Major 
4. Confirmed diagnosis of urethritis, proctitis or pharyngitis Ng – 
symptomatic or asymptomatic (Diagnosis of Ng will be by a positive 
NAAT performed according to the ITMs current laboratory 
protocols or for patients with urethritis a positive gram/methylene 
blue stain) 
Major 
Exclusion criteria 
 
5. Use of any macrolide antibiotics (azithromycin, clarithromycin, 
erythromycin, roxithromycin, spiramycin) in the previous 6 months 
Major 
6. Known contra-indications or allergy to ceftriaxone, azithromycin or 
lidocaine 
Major 


 
 
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7. Presence of any other condition, including other STIs that will 
(likely) require the administration of another antibiotic at the time 
of enrolment, as assessed by the treating physician 
Major 
Treatment violations 
 
1. Not following the randomized intervention 
Major 
Follow-up violations 
 
1. Visits performed outside the specified time window (day 13-15) 
Major 
Sampling violations 
 
1. Did not provide any samples 
Major 
 
 
5.2. Outcomes 
 
Primary outcome  
During the bioinformatic analyses, each identified antibiotic-resistance determinant 
will be categorized at the class level using a read-based classification tool. The primary 
outcome will be the ratio of mean macrolide resistance determinants in the day 14 
anorectal samples between the two treatment groups. This ratio will be calculated by 
dividing the mean normalized read count of macrolide-resistance determinants 
categorized at the class level in the CRO/AZM group by the corresponding mean 
quantity in the CRO group. 
 
Secondary outcomes 
The ratio of mean normalized read count of resistance determinants for each non-
macrolide antibiotic class in the day 14 anorectal samples between the two treatment 
groups 
 
Exploratory outcomes 
The mean (normalized) resistance determinants per treatment arm on days 0 and 14.  
 
Safety outcome 
The number of participants with (severe) adverse events in each treatment group. Since 
the IMPs are already marketed and widely used, a predefined list of AE (as listed below) will 
be reviewed at every study visit and reported in the source documents. These events will also 
be recorded in the eCRF (electronic Case Report Form):  
 
- 
Diarrhea (CRO, AZM) 
- 
Skin rash (CRO) 
- 
Headache (CRO, AZM) 
- 
Dizziness (CRO, AZM) 
- 
Nausea or vomiting (CRO, AZM) 
- 
Abdominal pain (AZM) 
- 
Pruritus (CRO) 
- 
Pain at injection site (CRO) 
 
 
 
 


 
 
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6. Statistical Methods 
 
6.1. Primary Efficacy Objective 
 
The primary analysis of assessing the ratio between the mean normalized read count 
of macrolide resistance determinants in anorectal microbiome between the two groups 
will be done using permutation test. The p-value of the test will determine if there is a 
statistically significant difference between the two means at a 5% significance level. 
The estimated ratio of the two arms is presented with a 95% confidence interval. As an 
alternative analysis, we will use (zero-inflated) negative binomial or Poisson models 
with the number of resistant determinants as the outcome, the allocation arm as the 
only effect and the logarithm of the number of bacterial load as the offset term.  
 
6.2. Secondary Efficacy Objectives 
 
The secondary objectives regarding mean comparisons will be done similarly to the 
primary analysis. Alternatively, a t-test in the log-transformed means of the two groups 
will be performed as a comparison to the permutation test. A 95% CI for the ratio of 
the two groups will be calculated using permutation testing.  
 
6.3. Exploratory Efficacy Objectives 
Analysis of covariance will be used to compare the results from day 14 adjusting for 
baseline results between the two treatment arms. Since the sample size is small, the 
individual difference scores will be calculated by subtracting the normalized resistance 
determinants of baseline from the ones of day 14. The two groups will, then, be 
compared using a two sample t-test.  
 
6.4. Safety Objectives 
 
The patient count with adverse events in the two groups will be compared using Fisher’s 
exact test.  
 
6.5. Other aspects 
 
a. Subgroup analyses 
No a priori subgroup analyses are defined. 
 
b. Multiplicity 
 
As this is a study with a single primary efficacy endpoint. For the non-macrolide 
comparisons, the significance level of the p-values will be adjusted using the 
Benjamini-Hochberg procedure.  
 
c. Interim analyses 
No interim analyses are planned. 
 
d. Missing data 
 
We estimate that 5% of the day 14 visits will be missed. These individuals will be 
dropped from the analyses, but the amount of missing data in each characteristic will 
be described.