The Diagnosis and Treatment of Chlamydia Trachomatis and Neisseria Gonorrhoeae in Pregnant Women to Prevent Adverse Neonatal Consequences.
Phase: NA
Conditions: Chlamydia Trachomatis Infection, Neonatal Infection, Preterm Birth, Gonorrhea
Sponsor: University of Southern California

Title: The diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae in pregnant 
women to prevent adverse neonatal consequences.” 
 
 
 
19 Sept 2022 
1
Title: The diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae in pregnant 
women to prevent adverse neonatal consequences. 
Protocol Version 7 
19 September 2022 
 
Co-Investigators 
1. Chelsea Morroni, MBChB, DTM&H, DFSRH, PhD; Senior Research Associate, Botswana 
Harvard Aids Institute Partnership; Botswana-UPenn Partnership.Tel: (267) 355-4855. Email: 
chelseaamorroni@gmail.com 
a. Doreen Ramogola-Masire, MD; Doreen Ramogola-Masire, BMedSci, BMBS, MRCOG, 
FCOG; Associate professor of Obstetrics and Gynaecology, and Deputy Dean for 
research and graduate studies at the University of Botswana, Faculty of Medicine. Tel: 
(267) 3554855. Email: doreen.masire@gmail.com 
b. Loeto Mazhani, MD; Paediatrician & former Head of Paediatrics, University of Botswana. 
Tel: (267) 71677145Email: lomazhani@yahoo.com 
c. Adriane Wynn, MPP, PhD; Assistant Professor, University of California, San Diego. Tel: 
+1 (916) 662-1497. Email: awynn@ucsd.edu 
d. Jeffrey D. Klausner, MD, MPH; Professor of Medicine & Public Health, University of 
California, Los Angeles. Phone: +1 (310) 794-3327. Email: 
JDKlausner@mednet.ucla.edu 
 
Key Personnel 
 
a. Aamirah Mussa, MPH; Programme Coordinator and Research Fellow, Botswana Harvard AIDS 
Institute Partnership. 
b. David Lawrence, MBChB MSc MRCP(UK); Associate Professor and Clinical Research 
Physician, London School of Hygiene and Tropical Medicine, Botswana Harvard AIDS Institute 
Partnership.  
c. Merrian Brooks, DO, MS; Global Health Fellow, Children’s Hospital of Philadelphia, Botswana-
UPenn Partnership.  
d. Rebecca Luckett, MD, MPH; Assistant Programme Director, Department of Obstetrics and 
Gynaecology, University of Botswana.  
e. Rebecca Zash, MD, BA; Research Associate, Harvard TH Chan School of Public Health.  
f. 
Rebecca Ryan, BMBCh MRCP DTM&H DFSRH PGCert Med Ed, Research Physician, 
Botswana Harvard AIDS Institute Partnership. 
g. Neo Moshashane, BSc (Hons); Research Assistant, Botswana Harvard AIDS Institute 
Partnership.  
h. Kehumile Ramontshonyana, BA; Research Assistant, Botswana Harvard AIDS Institute 
Partnership.  
i. 
Bame Bame, Research Nurse, Botswana Harvard AIDS Institute Partnership.  
j. 
Badani Moreri-Ntshabele, MBChB (UCT), FCOG (SA); Lecturer, Department of Obstetrics and 
Gynaecology, University of Botswana.  
k. Bogadi Loabile, MD/MPH, Infectious Disease Fellow, University of Pennsylvania.  
l. 
Neo Ndlovu; Research Assistant, Botswana Harvard AIDS Institute Partnership.  
m. Lefhela Tamuthiba; Research Assistant, Botswana Harvard AIDS Institute Partnership.  
n. Selebaleng Simon; Nurse Coordinator, Botswana Harvard AIDS Institute Partnership 

Title: The diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae in pregnant 
women to prevent adverse neonatal consequences.” 
 
 
 
19 Sept 2022 
2
o. Emily Hansman, UCGHI Fogarty GloCal Fellow, University of California Los Angeles 
 
 
Protocol Contents 
 
1. Issue 
2. Specific Aims 
3. Study Design 
4. Background and Significance 
5. Characteristics of the Subject Population 
6. Subject Identification and Recruitment 
7. Methods and Procedures applied to human subjects  
8. Research involving survey, questionnaires 
9. FDA approval (e.g. for experimental drugs, etc.) 
10. Data collection, Storage and Confidentiality 
11. Risk/Benefit Assessment  
12. Therapeutic Alternatives 
13. Financial Considerations  
14. Emergency Care and Compensation for Research-Related Injury 
15. Informed Consent 
16. Process of Consent 
17. Comprehension of the Information Provided 
18. Information withheld from Subjects 
19. Consent/Assent forms 
20. Conflicts of Interest 
21. Study Timeline 
22. Dissemination Plan 
 
1. Issue:   
Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and syphilis (caused by Treponema 
pallidum) are bacterial sexually transmitted infections (STIs) and are neglected causes of adverse 
neonatal outcomes. According to WHO modelling estimates, CT, NG, and syphilis are among the most 
common STIs globally. These infections are even more common among people living with HIV. The risk 
of vertical transmission during delivery is about 50%. HIV-infected pregnant women with CT, NG, 
and/or syphilis infections are at increased risk of MTCT of HIV. Other studies on CT and NG infections 
in neonates are over 20 years old and those infections are not systematically measured in sub-Saharan 
Africa and are largely unknown. 
  
Few countries provide routine antenatal testing for CT and NG. In most countries, including 
Botswana, the syndromic approach is used, which utilizes an algorithm to classify symptoms into STI 
syndromes and provide standardized treatment. Syndromic management lacks specificity and causes 
pregnant women to be unnecessarily exposed to antibiotics, and has low sensitivity, missing 
asymptomatic infections, putting neonates at risk. A study in Botswana found that among women with 
confirmed CT, NG, Trichomonas vaginalis (TV), only 10% reported vaginal discharge and 8% reported 
lower abdominal pain. In our pilot study, we found that less than 40% of women with etiologically 
diagnosed CT, NG, and TV infections reported having vaginal discharge, lower abdominal pain, and/or 

Title: The diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae in pregnant 
women to prevent adverse neonatal consequences.” 
 
 
 
19 Sept 2022 
3
painful urination. Thus, it’s possible that many infections are being missed among pregnant women in 
Botswana. While testing for syphilis is a routine part of antenatal care in Botswana, previous studies 
have shown that testing coverage and receipt of treatment are suboptimal. 
Our proposal has four specific aims. Aim 1: We will determine the burden of CT and NG 
infections and correlates of infection among asymptomatic pregnant women in Gaborone, Botswana by 
a) using diagnostic testing to estimate the prevalence of CT and NG infections at three time points: 1) 
first antenatal care visit, 2) third trimester antenatal visit, and 3) postnatal visit. We will also estimate the 
incidence of CT/NG infections between those visits and b) assess the correlates of infection. Aim 2: 
Will compare longitudinal neonatal outcomes for pregnant women tested for CT and NG infections 
during antenatal care with women who received standard antenatal care by a) estimating the frequency 
of vertical transmission of CT and NG infections and neonatal outcomes and the association with 
testing and treatment; and b) assessing independent factors that may be predictive of adverse neonatal 
outcomes. Aim 3: We will assess the burden of infection and markers of inflammatory response to CT 
and NG infection during pregnancy and associations with vertical transmission of CT and NG by a) 
testing immunologic factors associated with vertical transmission of CT and NG; b) determining the 
association between the Xpert® CT/NG assay’s pathogen-specific cycle threshold value (Ct) and 
transmission; and c) evaluating the frequency and distribution of Sample Adequacy Control (SAC) cycle 
threshold values (Ct) from the Xpert® CT/NG assay, and evaluate any correlations with the 
transmission of CT/NG to neonates. Aim 4: We will determine the acceptability, feasibility, and costs of 
implementing point-of-care (PoC) syphilis testing and treatment among pregnant women in Gaborone, 
Botswana by offering: 1) dual HIV and syphilis PoC testing for pregnant women who are HIV 
uninfected/undiagnosed and 2) PoC syphilis testing for pregnant women with a known HIV infection 
with same-day treatment.  
We will provide evidence to help evaluate the effects of testing on vertical CT/NG transmission 
and clinically important neonatal health outcomes, and to evaluate and understand biological correlates 
of CT/NG transmission.  
 
2. Specific Aims: 
a. Specific Aim 1:  To determine the burden of CT, NG infections and correlates of infection 
among asymptomatic pregnant women in Gaborone, Botswana. 
i. 1(a): Use diagnostic testing to estimate the prevalence of CT/NG infections at the 1st 
antenatal care visit, a 3rd trimester visit, and the 1st postnatal care visit; and 
estimate the incidence of infections between those visits. 
ii. 1(b): To assess the correlates of infection, including sociodemographic and 
behavioral factors (e.g. alcohol use), obstetric and medical information (e.g. HIV 
status), and sex partner characteristics.  
b. Specific Aim 2: To compare longitudinal neonatal outcomes for pregnant women tested & 
treated for CT/NG infections during antenatal care with women who received standard 
antenatal care. 
i. 2(a):  To estimate the frequency of vertical transmission of CT/NG infections and 
subsequent neonatal outcomes (e.g. ophthalmia neonatorum) and the association 
with maternal testing and treatment during antenatal care. 
ii. 2(b): To assess independent factors that may be predictive of adverse neonatal 
outcomes and control for these factors in the analysis. Exposure measures will 
include maternal sociodemographic and behavioral factors (e.g. alcohol use), 
obstetric and medical information (e.g. HIV status), partner characteristics, and 
adverse perinatal outcomes (e.g. low birth weight). 

Title: The diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae in pregnant 
women to prevent adverse neonatal consequences.” 
 
 
 
19 Sept 2022 
4
c. Specific Aim 3: To assess the burden of infection and markers of inflammatory response to 
CT/NG infection during pregnancy and associations with vertical transmission of CT/NG. 
i. 3(a): To test immunologic factors associated with vertical transmission of CT/NG by 
describing concentrations of genital inflammatory cytokines in participants who test 
positive for CT/NG.  
ii. 3(b): To determine the association between the Xpert® CT/NG assay’s pathogen-
specific cycle threshold value (Ct), a possible marker for burden of infection, and 
vertical transmission of CT/NG to neonates.  
iii. 3©: To evaluate the frequency and distribution of Sample Adequacy Control (SAC) 
cycle threshold values (Ct) from the Xpert® CT/NG assay, which are a measure of 
tissue inflammation, and evaluate any correlations with the transmission of CT/NG to 
neonates. 
d. Specific Aim 4: To determine the acceptability, feasibility, and costs of implementing point-
of-care (PoC) syphilis testing and treatment among pregnant women in Gaborone, 
Botswana.  
i. 4(a) To assess the acceptability and feasibility of PoC syphilis testing and treatment. 
(Dual HIV and syphilis PoC testing will be offered to pregnant women who are HIV 
uninfected/undiagnosed and rapid syphilis testing for pregnant women with a known 
HIV infection). Our primary 
outcome will be defined as 
acceptability of testing, 
which is the proportion of 
women tested out of all 
those eligible and seeking 
care in the study clinics. 
Secondarily, we will 
assess the proportion of 
women who received 
treatment among those 
with a reactive syphilis test 
result. We will assess any 
treatment, treatment 
completion, and time to 
treatment. 
ii. To evaluate the unit costs 
and patient costs 
associated with antenatal 
PoC syphilis testing and treatment compared to the standard of care the standard of 
care. 
 
3. Study Design 
Specific Aim 1:  To determine the burden of CT and NG infections and correlates of infection 
among asymptomatic pregnant women in Gaborone, Botswana. 
Flow diagram 

Title: The diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae in pregnant 
women to prevent adverse neonatal consequences.” 
 
 
 
19 Sept 2022 
5
 
Methods and Procedures: To accomplish Aim 1, we will conduct a cohort study among antenatal 
patients at collaborating Greater Gaborone District Health Management Team (DHMT) clinic sites (STI 
testing clinics). First, 1(a): Use diagnostic testing to estimate the prevalence of CT/NG infections at the 
1st antenatal care visit, a 3rd trimester visit, and the 1st postnatal care visit; and estimate the incidence 
of infections between visits. 1(b): To assess the correlates of infection, including sociodemographic and 
behavioral factors (e.g. alcohol use), obstetric and medical information (e.g. HIV status), sex partner 
characteristics, and partner notification and treatment outcomes. 
 
Recruitment and Eligibility: We will recruit 250 study participants from pregnant women presenting 
for antenatal care services at designated study ‘testing’ clinics in Gaborone. Eligibility/Inclusion criteria 
(also described in Section E) include: 1) Age ≥ 15 years, 2) Currently pregnant, 3) Attending first ANC 
visit, 4) 27 weeks of gestation or less, 5) Not currently experiencing CT/NG-related symptoms 
(determined by validated screening tool), 6) Not treated for CT/NG in the past 30 days 7) Willingness to 
provide self-collected specimens for CT/NG testing (at three time points), 8) Willingness to return for a 
test of cure (if test positive during antenatal care), 9) Will reside in Gaborone through the time of 
delivery and 1st postnatal visit, 10) Willingness to have neonates tested for CT/NG at their first post-
natal visit, And 11) Mentally competent to understand the informed consent.  
Patients will be screened for eligibility via obstetric chart review at the time of the appointment. 
Those who are ineligible due to STI-related symptoms will be flagged and identified for healthcare 
providers to assess for syndromic management (standard care). All eligible patients will be provided 
information about CT and NG infections and study risks and benefits. Those providing informed 
consent will be enrolled, instructed on how to self-collect a CT/NG specimen and asked to share 
several forms of detailed contact information to assure follow-up. Reasons for ineligibility will be logged 
with age, gestational age, and reason for ineligibility, to assess generalizability. Similar data and 
reasons for decline will also be recorded.  
A sample of up to 20 sex partners will be recruited for participation in a brief semi-structured 
interview. Partners will be recruited through the following channels a) snowball recruitment via study 
participants who give informed consent to contact their partner(s) b) sex partners presenting to a study 
clinic for STI treatment using a contact slip c) sex partners contacting the study team via contact 
information provided with EPT materials d) partners attending antenatal or postnatal care visits with 
partners. Eligibility criteria include 1) Age ≥ 15 years 2) Sex partner of a pregnant women being treated 
for CT, NG, Syphilis, or an STI syndrome 3) Willing to complete a recorded semi-structured interview 
and 4) Mentally competent to understand the informed consent.  
A sample of up to 20 stakeholders will be recruited for participation in a brief semi-structured 
interview. Eligibility criteria include 1) a stakeholder, defined as a clinician who provides antenatal or 
sexual and reproductive healthcare, OR a program manager at a clinic that provides sexual and 
reproductive healthcare, OR a policymaker in the field of maternal and sexual and reproductive health 
and 2) Willing to complete a recorded semi-structured interview. 
 
Specimen Collection, Transport, Processing and Storage: Specimens will be labeled with a unique 
study ID by trained study staff, who will place them in secure storage for up to 24 hours at 2° - 30°C 
until testing.  
 
Laboratory Testing:  CT /NG testing: will be performed using Xpert® CT/NG assay [Cepheid, 
Sunnyvale, CA] by trained staff. All specimens will be run on-site with standard controls, per 
manufacturer’s instructions. (See Aim 3 for quality control). (Note: The Xpert is currently being used in 
Botswana for TB testing).  
 

Title: The diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae in pregnant 
women to prevent adverse neonatal consequences.” 
 
 
 
19 Sept 2022 
6
Reporting and Treatment: Antenatal CT and NG testing will be conducted at enrollment (1st ANC), 
during the third trimester, and at the 1st postnatal visit (See Figure 1). Results and treatment will be 
provided in the same way at each time period. Results: All participants will receive results on the same 
day as testing (in person or by phone). Treatment: Appropriate clinical staff will provide treatment per 
the Botswana MoH STI treatment protocols. Treated participants for CT/NG will be asked to provide a 
second specimen at least 4 weeks after treatment to document treatment outcome (test of cure). Test 
results will be recorded by study staff in the patients’ treatment record files as well as into the study 
dataform. Partner Services: All study staff will be trained on appropriate and effective methods for 
counselling women on how to communicate test results to partner(s) in a nonthreatening and blameless 
manner. (For example, it’s important to clarify that the duration of sexually transmitted infections during 
pregnancy is unknown, many infections are asymptomatic, and infections do not have implications 
about partner fidelity). Additionally, participants will be given several options for partner services: 1) No 
disclosure: In the case of risk for intimate partner violence (IPV). 2) Supportive disclosure: where the 
participant is assisted by trained study staff either in person or over the phone in notifying their 
partners. Also, participants will be given the option of bringing partners to the study clinics for 
counselling and treatment. 3) Expedited partner therapy for CT/NG treatment: where the participant can 
bring treatment to partners prior to examination by a healthcare provider. IPV: will be proactively 
assessed and managed for all participants, not only those testing STI positive. To that end, we will use 
a validated screening tool adapted from Rabin RF et al.[1] to assess participant risk for intimate violence 
and other negative partner responses to notification of an STI. Based on the screening results, when 
appropriate, IPV risk will be mitigated by referring women to local support services available in the 
country for free counseling and support, the NGO Botswana Family Welfare Association (BOFWA; 
http://www.bofwa.org.bw/). We will also provide a 24-hour emergency contact line through which 
participants can receive information and support and referrals in general. 
 
Data collection: Participants will be asked to respond to a survey on socioeconomic 
characteristics, relationship status and sexual history (e.g. number of condomless sex acts), other risk 
behaviors (e.g. alcohol use), obstetric/medical history, and prior STI symptoms and treatment in the 
past year. A sub-set of participants will be asked additional questions on partner notification and 
treatment experiences postnatally. A data collection instrument will be used to abstract obstetric, and 
medical information from the patient-held and clinic records, including receipt of syphilis testing, results 
and titre, and treatment.  
The sample of sex partners will participate in a brief semi-structured interview on partner notification 
and treatment experiences. The sample of stakeholders will participate in a brief semi-structured 
interview on antenatal diagnostic testing for chlamydia and gonorrhea, point-of-care syphilis testing, 
and STI expedited partner therapy. 
 
Specific Aim 2: To compare longitudinal neonatal outcomes for pregnant women tested & 
treated for CT/NG infections with women who received standard antenatal care.
 
 
Methods and Procedures: 
Simultaneously with Aim 1, 250 antenatal patients at designated study 
‘standard of care’ clinics will be enrolled as a comparison group receiving the standard care with 
additional partner services. At standard of care clinics, CT/NG testing will be offered to mothers and 
neonates at the 1st postnatal visit for all 500 participants to accomplish 2(a): To estimate the frequency 
of vertical transmission of CT/NG infections and adverse neonatal outcomes and association with 
maternal testing and treatment. And subaim 2(b): To assess independent factors associated with 
adverse neonatal outcomes and control for these factors in the analysis. Exposure measures will 
include maternal sociodemographic, behavioral, obstetric, and medical information, and partner 
treatment, and adverse perinatal outcomes (e.g. low birth weight). 
 

Title: The diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae in pregnant 
women to prevent adverse neonatal consequences.” 
 
 
 
19 Sept 2022 
7
Recruitment/Eligibility:
 Women will be similarly recruited and enrolled, and eligibility criteria are 
identical to those in the STI testing group (see Aim 1). Those who are ineligible due to STI-related 
symptoms will be flagged and identified for healthcare providers to assess for syndromic management 
(standard care). All eligible patients will be invited to participate, and those providing informed consent 
will be enrolled. The comparison group will receive the standard of care for management of STIs during 
antenatal care with additional IPV screening and partner services offered to those in Aim 1. As in Aim 1, 
a data collection instrument will be used to collect sociodemographic and behavioral factors, and 
obstetric/medical information, and data will be abstracted from the patient-held and clinic records. All 
mothers will be provided with informed consent for their neonate’s participation at enrollment. 
Postdelivery, study staff will provide an updated verbal consent to mothers to collect and tests neonatal 
specimens. 
 
Laboratory testing:
 All participants will test for CT/NG infection using the Xpert® CT/NG assay at the 
first postnatal visit using self-collected swabs. Women testing positive will receive antimicrobial 
treatment with options for partner treatment described in Aim 1. For neonates of mothers testing 
positive for CT/NG, the study follow-up nurse will collect ocular and nasopharyngeal swab samples for 
testing of CT/NG transmission using the Xpert® CT/NG assay. Studies have demonstrated the efficacy 
of PCR tests for infant diagnosis using ocular and nasopharyngeal swabs.[2, 3] Infants testing positive 
will receive treatment according to Botswana guidelines (20mg/kg of azithromycin (liquid formulation) 
for CT, injection of 50mg/kg of ceftriaxone for NG) and referrals.[4] Efforts will be made to provide same-
day results.  
 
1st Postnatal Visit Survey : Primary outcome will be collected by the study follow-up nurse and include: 
CT/NG test result from neonate ocular/nasopharyngeal swabs. Secondary outcomes include clinical 
evidence or records of conjunctivitis and history of pneumonia identified in interviews or medical charts; 
and inpatient length of stay. Data on potential confounders will be collected from three sources: 1) 
Interviewer-administered survey, AND 2) Patient obstetric record, OR 3) Hospital/clinic records. (Study 
staff have experience with all 3 sources). If new neonatal conditions are identified, the nurse will ensure 
necessary referrals. 
 
Retention and Follow -up: Multiple forms of contact information will be collected at enrollment for all 
participants to ensure retention. All study visits will take place during regularly scheduled antenatal and 
postnatal care visits. Participants will be requested to give permission for communication through text 
messages, which will remind about upcoming visits. Participants who do not return for scheduled visits 
will be actively contacted by both clinic and study staff and encouraged to return for care. The study 
team will follow-up with all participants weekly around the expected date of delivery and participants will 
be encouraged to notify the team of first postnatal visit. Ten attempts will be made through various 
contact methods.  
 
Specific Aim 3: To assess markers of inflammatory response to CT/NG infection during 
pregnancy and associations with vertical transmission of CT/NG.
 
 
Methods and Procedures:
  At the baseline, third trimester, and 1st postnatal care visit, self-collected 
vaginal swabs[5] and EDTA plasma samples will be collected from all participants in the STI testing 
group, and used to measure cytokine concentrations. Cytokines will be identified using the Luminex 
xMAP® 48-plex assay method located at Stanford University in order to accomplish 3(a): To test 
immunologic factors associated with vertical CT/NG transmission by describing concentrations of 
inflammatory cytokines in participants who tested positive for CT/NG. A secondary analysis of data 
collected during maternal CT/NG testing at the first postnatal visit (Aim 2) will be performed to 
accomplish 3(b): To determine the association between the Xpert® CT/NG assay’s pathogen-specific 
cycle threshold value (Ct), which is a marker for the burden of infection, and vertical transmission of 

Title: The diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae in pregnant 
women to prevent adverse neonatal consequences.” 
 
 
 
19 Sept 2022 
8
CT/NG to neonates; and 3(c): To assess the frequency and distribution of Sample Adequacy Control 
(SAC) cycle threshold (Ct) values from the Xpert® CT/NG assay, which may be a marker of 
inflammation, and evaluate correlations with the transmission of CT/NG to neonates. 
 
Recruitment/Eligibility:
 Eligibility criteria for the comparison group are identical to those in Aims 1 and 
2. Data will only be collected from participants in the STI testing group. 
 
Specimen Collection, Transport, Processing and Storage: 
A self-collected swab for cytokine 
measurement will be collected at the baseline, third trimester, and first postnatal care visit and stored at 
-80C. A peripheral blood sample (2-4mL) will be collected at the baseline, third trimester, and postnatal 
study visits. The blood sample will be processed and aliquots of EDTA plasma will be stored at -80C. 
The swabs and plasma samples will be transported in dry ice to Stanford University for processing. 
Luminex will be used to measure the concentrations of 48 cytokines in the 48-plex assay (including IL-
1α, IL-1β, IL-6, IL-7, IL-8, IL-10, granulocyte macrophage colony- stimulating factor [GM-CSF], 
interferon-γ inducible protein [IP]-10, monocyte chemoattractant protein [MCP]-1, MIP-1α, MIP-1β, and 
tumor necrosis factor [TNF]-α). These cytokines were selected based on previously identified 
associations with genital infection.[6] Additionally the pathogen-specific and SAC Ct results will be 
automatically generated for each self-collected vaginal sample tested using the Xpert® CT/NG assay at 
delivery. 
 
Laboratory Testing : Once the specimen is collected, study staff will follow instructions for sample 
preparation and testing. Luminex assays enable fast and accurate cytokine measurements by utilizing 
specially prepared microspheres, internally dyed with a graded mixture of red or infrared fluorescent 
dyes. Varying the degree to which the beads are internally dyed creates hundreds of different 
fluorescent profiles that can be individually interrogated and classified in a single sample.[7] The BHP 
lab in Gaborone can conduct cytokine analysis using an ELISA quantitative; however, this assay is 
currently limited to only IL-6, ICAM1, and VCAM. Further, the ELISA is a single assay and sample 
processing would be costly and time-consuming compared to the Luminex multiplex assay. 
 
Specific Aim 4:  To determine the acceptability, feasibility, and costs of implementing point-of-care 
(PoC) syphilis testing and treatment among pregnant women in Botswana by offering: 1) dual HIV and 
syphilis PoC testing for pregnant women who are HIV uninfected/undiagnosed and 2) PoC syphilis 
testing for pregnant women with a known HIV infection with same-day treatment to women in the 
designated PoC clinic. PoC testing will be offered in addition to routine syphilis and HIV testing. 
 
Methods and Procedures: 
An additional 200 antenatal patients at the designated PoC clinic and 200 
at the ‘standard of care’ clinic will be enrolled (400 total). In the PoC clinics, participants who are HIV 
uninfected/ undiagnosed will be offered PoC dual HIV/syphilis testing and women living with HIV will be 
offered a PoC syphilis test. PoC participants will receive same-day results and treatment. Participants 
in the standard of care clinic will continue to receive the usual care.  
 
Additionally, analysis will incorporate the data from 200 participants previously enrolled in the CT/NG 
parent study. These data will be used as a “pre-test” and will include all 100 women enrolled in the 
standard of care clinic and all 100 women enrolled in the PoC testing clinic.  
 
Recruitment and Eligibility: Recruitment will be the same as Aim 1 and the risks and benefits of PoC 
syphilis testing will be explained. Reasons for declining to participate will be recorded as well as basic 
information, including age and gestational age to assess generalizability. Eligibility criteria will be the 
same as Aim 1, after removing CT/NG testing-specific criteria, and will include: 1) Age ≥ 15 years, 2) 
Currently pregnant, 3) Attending first ANC visit, 4) 27 weeks of gestation or less, 5) Will reside in 

Title: The diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae in pregnant 
women to prevent adverse neonatal consequences.” 
 
 
 
19 Sept 2022 
9
Gaborone through the time of delivery and 1st postnatal visit, And 6) Mentally competent to understand 
the informed consent. 
 
Laboratory Testing: PoC syphilis: Participants who are HIV-uninfected/undiagnosed in the PoC clinics 
will receive a test using the SD BIOLINE HIV/Syphilis Duo test, which is a solid phase 
immunochromatographic assay for the qualitative detection of antibodies to all isotypes(IgG, IgM, and 
IgA) specific to HIV-1/2 and/or Treponema pallidum (TP) simultaneously in human serum, plasma, or 
whole blood. The WHO prequalification performance evaluation found a final sensitivity for HIV was 
100% (95% CI: 98.2-100%) and specificity of 99.5% (95% CI: 97.2-100%). The final sensitivity for 
antibodies to treponema pallidum was 87% (95% CI: 81.5%-91.3%) and the specificity was 87% (95% 
CI: 97.2-100%).[8] As per Botswana guidelines, HIV will be diagnosed with two serial HIV reactive test 
results (Dual PoC followed by rapid HIV test). For participants who are living with HIV and seeking care 
in the PoC clinics, we will offer a rapid, PoC blood test for syphilis with WHO prequalification. All 
participants will continue to receive the standard of care, non-treponemal detecting IVD (RPR/VDRL) 
test. Results from the RPR/VDRL will be used to confirm test results and differentiate individuals with 
active (untreated) infection from those who have been successfully treated for past infection.[9] 
Reporting and Treatment: PoC Results: All participants will receive results in person, on the same day 
as testing. Treatment: Appropriate clinical staff will provide treatment per the Botswana MoH STI 
treatment protocols (see Treatment section below). Test results will be recorded in the participant 
obstetric record, clinic records, as well as into the study data form. Partner Services will be offered in 
the same manner as Aim 1; however, expedited partner treatment will not be an option for syphilis 
treatment. Standard of Care: Participants will continue to receive syphilis testing per Botswana national 
guidelines, which includes a VDRL/RPR test at the first ANC visit with delayed results and treatment. 
Typically, blood specimens are sent to a central lab for processing and results are provided to clinic 
staff after at least three days. Women are called to return to the clinic for receipt of results. Test results 
and treatment are recorded in the patient-held and clinic records. 
 
Data collection: For participants in the PoC clinic, acceptability of PoC syphilis testing and receipt of 
treatment (with dates) will be recorded by study staff in study documents, and the patient-held and 
clinic records. Participants who decline and reasons for decline will be recorded in study records. For 
participants in the standard of care clinic, the patient-held and clinic obstetric records will be reviewed 
to identify receipt and dates of syphilis testing and treatment. We will also abstract obstetric and health 
information (para, gravida, gestational age, HIV status, antiretroviral treatment) from the records of all 
participants. At the time of enrollment, participants will be asked to respond to a short interviewer-
administered questionnaire to collect basic sociodemographic information and costs incurred related to 
syphilis testing. Participants will also respond to another short survey on costs during their third 
trimester, which will be conducted over the phone. Cost-related questions will inquire about travel, 
overnight lodging, out-of-pocket care costs; and the opportunity costs associated with travel, wait, and 
appointment times. Participants testing positive will be eligible to participate in a brief semi-structured 
interview on partner notification and treatment.  
 
Outcomes: Our primary outcome is acceptability of syphilis testing at the first ANC visit (proportion 
tested for syphilis out of all eligible women receiving care in the study clinics during implementation of 
the study). For participants in the PoC clinic, acceptability will be defined as having a PoC test with 
recorded results. Those who decline PoC testing will be identified as not accepting and will be included 
in the denominator. For those in the standard of care, the numerator will be women who receive an 
RPR/VDRL syphilis test recorded in patient-held or clinic records. The denominator for both groups will 
be all women attending the study clinic during implementation and eligible for participation (1. Age ≥ 15 
years, 2. Currently pregnant, 3. Attending first ANC visit, 4. 27 weeks of gestation or less, 5. Will reside 

Title: The diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae in pregnant 
women to prevent adverse neonatal consequences.” 
 
 
 
19 Sept 2022 
10
in Gaborone through the time of delivery and 1st postnatal visit, And 6. Mentally competent to 
understand the informed consent).  
 
Additional outcomes will be related to treatment and costs. We will report the proportion of women who 
received treatment among those with a reactive syphilis test result. We will assess differences in any 
treatment, treatment completion, and time to treatment.  
 
The primary cost outcome is the average unit cost per woman tested for each strategy and will include 
both patient-related costs and healthcare system costs. Healthcare-related cost data will be collected 
from in-country data sources. Patient data will be collected from the short survey. 
 
Analysis: First, comparisons will be made across four groups of participant data: 1) participants in the 
standard of care clinic enrolled as part of the CT/NG study prior to implementation of the syphilis testing 
sub-study called “Standard of Care pretest” (n=100); 2) participants in the standard of care clinic 
enrolled as part of the syphilis testing sub-study called “Standard of Care post-test” (n=200); 3) 
participants in the PoC clinic enrolled as part of the CT/NG study prior to implementation of the syphilis 
testing sub-study called “PoC pretest” (n=100); and 4) participants in the PoC clinic enrolled as part of 
the syphilis testing sub-study called “PoC post-test” (n=200).  
 
To assess differences in receipt of syphilis testing, we will use a difference-in-difference (DiD) strategy, 
which takes the difference between two before and after differences (pre-post-test with 
comparison).[25] DiD will be performed using a logistic regression model with a variable for whether the 
individual was in the PoC clinic (PoC), whether or not it is the post period (Post), an interaction variable 
for the product of those two variables, and covariates. The primary outcome (Y) is the proportion of 
eligible women who are tested for syphilis at their first ANC visit: 
Y=a+bPoCPoC+bPostPost+bint(Post*PoC)+bcovariates  Models will also be stratified by HIV status to 
see if PoC had a differential impact among WLH. Variables for which there were significant differences 
between the intervention and comparison group will be included in multivariable model.  
 
Secondarily, will also assess receipt of treatment using DiD analysis by comparing the pre-test and 
post-test differences in terms of the proportions of participants who had a reactive test who: 1) received 
the first dose of treatment, and 2) completed treatment. Prevalence of syphilis will also be reported 
according to treatment group and HIV status as the proportion of individuals testing positive divided by 
the number of individuals tested. Exact binomial 95% confidence intervals for prevalence will be 
estimated. All analyses will be conducted with Stata 17.0 (Stata Corporation, College Station, TX). 
 
To compare differences in costs, recurrent costs will be calculated per test, and capital and training 
costs will be summed and divided by the number of women tested. Those costs will be added to get an 
average unit cost for each strategy. For the intervention strategy, we will also calculate the average 
cost by HIV status at first ANC (i.e. the cost of delivering the dual HIV/syphilis test and the rapid 
syphilis). We will compare the mean costs using the non-parametric Mann-Whitney U test.[10] For 
hypothesis 4, patient costs will be summed and an average patient cost will be calculated for each 
strategy, which will be compared using either the Mann-Whitney U test or Student’s t-test. Cost 
components will be examined to determine key cost drivers and to identify potential efficiencies that 
could be achieved in future scale-up.   
 
4. Background and Significance 
Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and syphilis (caused by Treponema 
pallidum) infections are bacterial STIs that cause adverse neonatal outcomes.[11-22] The risk of vertical 

Title: The diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae in pregnant 
women to prevent adverse neonatal consequences.” 
 
 
 
19 Sept 2022 
11
transmission of CT/NG during delivery is about 50%.[23] Among infants born to mothers with untreated 
CT infection, 30–50% develop clinical conjunctivitis and 10-20% develop pneumonia due to CT.[23, 24] 
Among infants born to mothers with untreated NG infection, the risk of conjunctival infection is up to 
48%, which can result in corneal damage and blindness.[25] A recent study in South Africa found that 
among infants born to a mother with CT, NG, or Trichomonas vaginalis (TV) infections, 37% had 
nasopharyngeal colonization with the same STI organism.[26] Other studies on CT/NG infections in 
neonates are over 20 years old.[25, 27, 28] HIV-infected pregnant women with CT/NG infections are at 
increased risk of MTCT of HIV.[29, 30] Untreated syphilis during pregnancy is associated with increased 
risk for early fetal death, stillbirth, preterm birth, low birthweight, neonatal death, and clinical disease in 
infants.[22] Congenital syphilis is the second leading cause of preventable stillbirth globally.[31] 
According to the WHO, CT, NG, and syphilis are the most common STIs globally with 131 
million new cases of CT, 78 million new cases of NG infections, and 6 million new cases of syphilis 
annually.[32] These infections are more common among people living with HIV.[33] Despite the large 
burden of CT/NG infections and the above mentioned adverse outcomes, few countries have guidelines 
that recommend routine antenatal CT/NG testing.[34] The WHO STI Treatment Guidelines are silent on 
the issue due to lack of evidence.[35] In most low/middle countries, syndromic management (SM) is 
used to manage CT/NG infections. SM utilizes an algorithm to classify symptoms and clinical signs into 
STI syndromes, and patients are treated with standardized drug regimens.[35, 36] SM lacks specificity, 
causing unnecessary exposure to antibiotics, and has low sensitivity, missing the large proportion of 
asymptomatic infections and potentially putting neonates at risk for transmission.[37-40] A study in 
Botswana found that among women with confirmed CT, NG, Trichomonas vaginalis (TV), only 10% 
reported vaginal discharge and 8% reported lower abdominal pain.[41] In our pilot study, we found that 
less than 40% of women with etiologically diagnosed CT, NG, and TV infections reported having 
vaginal discharge, lower abdominal pain, and/or painful urination.[33] Thus, it’s possible that many 
infections are being missed among pregnant women in Botswana. Botswana national guidelines 
recommend universal screening and treatment of syphilis during pregnancy, which is conducted using 
the rapid plasma reagin (RPR) test or the venereal disease research laboratory (VDRL) with delayed 
results and treatment. However, a national surveillance study reported that only 59% of those with a 
reactive RPR were treated,[42] and treatment was delayed by five weeks on average.[43] 
As accurate and rapid testing technology is increasingly affordable, more evidence on the 
benefits of antenatal CT/NG testing is needed. This study will utilize an innovative reverse transcription 
polymerase chain reaction (PCR) assay that can provide results within 90 minutes. Xpert® CT/NG also 
has high sensitivity (97.6% and 95.6% for CT/NG respectively) and specificity (99.4% and 99.8% for 
CT/NG respectively) and functions well in resource-constrained environments such as those proposed 
here.[44] For syphilis testing, we will use WHO pre-qualified dual syphilis/HIV point-of-tests and rapid 
syphilis tests (SD Bioline), which will be offered in addition to the standard of care syphilis screening 
procedure. 
The proposed study will estimate the prevalence and correlates of infection among pregnant 
women who present to antenatal care without STI-related symptoms, and thus would not be treated for 
infections through the standard care. We will assess the impact of antenatal CT/NG testing to prevent 
vertical transmission and associated adverse health outcomes compared to the standard of care. We 
will use markers of tissue inflammation specific to the Xpert® to explore the inflammatory responses to 
CT/NG and associations with vertical transmission. We will assess the acceptability, feasibility, and 
costs associated with PoC dual syphilis/HIV testing. We hope to inform stakeholders and policy makers 
as they work to improve management of STIs during antenatal care in order to improve maternal and 
neonatal outcomes. 
We will include women aged 15-17 years, who are pregnant and receiving care, because 
pregnancy and curable sexually transmitted infections (STIs), such as Chlamydia trachomatis, 
Neisseria gonorrhoeae and syphilis are common among adolescents in Botswana. Recent research by 
BHP determined that of 737 adolescent females, aged 16-19 years, 340 (46%) reported being sexually 

Title: The diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae in pregnant 
women to prevent adverse neonatal consequences.” 
 
 
 
19 Sept 2022 
12
active. Of these, 121 (36%) reported having at least one child. A 2007 study of STIs among pregnant 
women in Gaborone, found that age was a strong predictor of C. trachomatis and/or N. gonorrhoeae 
infections; and, in a sample of 703 pregnant women, the prevalence of cervical infections was highest 
among teenagers (22%; 95% CI: 13-32%).[41] Recent research in Southern Africa has similarly found 
that the prevalence of STIs was higher among younger women.[45] Thus, in order to understand the 
burden and address the increased risks for STIs faced by women under 18 years, more research is 
needed among this group.  
 
 
5. Characteristics of the Subject Population: 
a. Number of subjects / Sample size:  
Pregnant women attending their first ANC care visit at 27 weeks gestation or less. Participants must be 
15 years of age and not treated with azithromycin, doxycycline, cefixime, or ceftriaxone in the past 30 
days.  
 
Specific Aim 1 (STI testing group): We will recruit 250 study participants for CT and NG testing.  
 
Specific Aim 2 (Standard of care comparison group): We will recruit 250 antenatal patients from  
designated study ‘standard of care’ clinics who will be enrolled as a comparison group receiving the 
standard care with additional IPV and partner services and postnatal care testing (Total N=500). 
Among participants who test positive during postnatal care, we will offer infant testing (Estimated N=30 
infants). 
 
For Specific Aims 1 & 2, study staff will remain at study clinics during operating hours for the duration of 
the recruitment period. We will consecutively screen all pregnant women for eligibility and offer 
enrollment to all eligible women.  
 
Specific Aim 3 (Inflammatory markers): We will include women in the STI testing group (Estimated 
N=250). 
 
Specific Aim 4: We will enroll an additional 400 antenatal patients for the PoC syphilis sub-study (200 
will receive the standard of care and 200 will be offered PoC syphilis testing in addition to the standard 
of care) 
 
b. Inclusion/Exclusion Criteria: 
 
• Inclusion Criteria: 
o Age ≥ 15 years, 
o Currently pregnant,  
o Attending first ANC visit,  
o 27 weeks gestation or less 
o Not currently experiencing CT/NG-related symptoms (determined by validated screening 
tool),  
o Not treated for CT/NG in the past 30 days,  
o Willingness to provide self-collected specimens for CT/NG testing (for the STI-testing 
group, this will take place at their first ANC visit, at another visit in their third trimester, 

Title: The diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae in pregnant 
women to prevent adverse neonatal consequences.” 
 
 
 
19 Sept 2022 
13
and at postnatal care. For the standard of care group, samples will only be collected at 
postnatal care),  
o Willingness to return for a test of cure if CT/NG test is positive during antenatal care,  
o Will reside in Gaborone through the time of delivery and 1st postnatal visit,  
o Willingness to have neonates tested for CT/NG at their first postnatal visit, 
o Mentally competent to understand the informed consent. 
 
• Exclusion Criteria: 
o Not mentally competent to understand study procedures or give informed consent, 
o Individuals < 15 years, 
o Men, 
o Women who are not pregnant, 
o Pregnant women not attending their first antenatal visit, 
o Pregnant women at >27 weeks gestation 
o Pregnant women with current STI-related symptoms (will receive standard of care), 
o Treated for an STI in the past 30 days 
 
c. Determining Eligibility --- Study Screening: Screening of prospective participants is confidential 
and will be undertaken by study personnel with training in ethical treatment of human subjects.  
 
d. Reasons for Exclusion: 
This study is designed to investigate benefits to women and infants of testing asymptomatic pregnant 
women for Chlamydia trachomatis and Neisseria gonorrhoeae during their first and third antenatal care 
visit. Therefore, women who are not pregnant as well as men are excluded from participation. Women 
with symptoms will receive syndromic management and those treated in the last 30 days are likely to 
be cured of STIs. 
 
e. Vulnerable Subjects:  As this study is conducted in Botswana, all interviews and exchange of 
information is conducted in English or Setswana. As the study concerns antenatal and postnatal 
testing, participants are pregnant women and infants. 
 
f. Inclusion of women aged 15-17 years: As we are including participants under 18 years, we will add 
additional protections for minors such as: reporting requirements for abuse and referrals to social 
workers and added support for partner notification. We have also added an adolescent health expert to 
our team to advise on these issues.  
 
6. Subject Identification and Recruitment:  
Specific Aim 1: Patients will be screened for eligibility via obstetric chart review and brief screening 
questions at the time of the appointment. All eligible patients will be provided information about CT/NG 
infections and study risks and benefits. Those providing informed consent will be enrolled, instructed on 
how to self-collect a CT/NG specimen and asked to share several forms of detailed contact information 
to assure follow-up. Reasons for ineligibility will be logged with age, gestational age, and reason for 
ineligibility, to assess generalizability. Similar data and reasons for decline will also be recorded. A data 
collection instrument will be used to collect sociodemographic, behavioral, sex partner, obstetric, and 
medical information. Data will be abstracted both from patient medical records and interviews by study 
staff.  
The sample of sex partners will be recruited through the following channels a) snowball recruitment 
via study participants who give informed consent to contact their partner(s) b) sex partners presenting 

Title: The diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae in pregnant 
women to prevent adverse neonatal consequences.” 
 
 
 
19 Sept 2022 
14
to a study clinic for STI treatment using a contact slip c) sex partners contacting the study team via 
contact information included with EPT materials (with permission of index patient) d) partners attending 
antenatal or postnatal care visits with partners.  
The sample of stakeholders will be recruited through email, word of mouth and visits to study 
clinics. 
 
Specific Aim 2: Women in the Standard of Care group will be similarly recruited and enrolled, and 
eligibility criteria are identical to those in the STI testing group (see Aim 1). All eligible patients will be 
invited to participate, and those providing informed consent will be enrolled. The Standard of Care 
group will receive the standard of care in Botswana for the management of STIs with additional intimate 
partner violence (IPV) screening and partner services offered to those in Aim 1. As in Aim 1, a data 
collection instrument will be used to collect sociodemographic and behavioral factors, and 
obstetric/medical information. All mothers will be provided with informed consent for their neonate’s 
participation at enrollment. Postdelivery, study staff will provide an updated verbal consent to mothers 
to collect and tests neonatal specimens. 
 
Specific Aim 3 At the baseline, third trimester and postnatal care study visit, self- collected swabs [5] 
and plasma samples (2-4mL) will be collected from participants in the STI testing group, and used to 
measure cytokine concentrations. Cytokines will be identified using the Luminex xMAP® 48-plex assay 
method located at Stanford University in order to test immunologic factors associated with vertical 
CT/NG transmission by describing concentrations of inflammatory cytokines in participants who tested 
positive for CT/NG. We will also conduct a secondary analysis of data collected during maternal CT/NG 
testing at the first postnatal visit (Aim 2) will be performed to determine the association between the 
Xpert® CT/NG assay’s pathogen-specific cycle threshold value (Ct), which is a marker for the burden of 
infection, and vertical transmission of CT/NG to neonates; and assess the frequency and distribution of 
Sample Adequacy Control (SAC) cycle threshold (Ct) values from the Xpert® CT/NG assay, which may 
be a marker of inflammation, and evaluate correlations with the transmission of CT/NG to neonates. 
 
Specific Aim 4: Women in the syphilis sub-study will be similarly recruited and enrolled as in Aim 1 
and 2. All participants will receive the standard of care in Botswana for syphilis testing with additional 
IPV screening and partner services offered to those in Aims 1 and 2.  A simplified data collection 
instrument will be used to collect sociodemographic and behavioral factors, and obstetric/medical 
information with additional questions related to patient costs incurred due to syphilis care. Participants 
recruited into the PoC syphilis testing clinic will provide a blood sample for PoC syphilis testing and 
treatment.  
 
7. Methods and Procedures applied to human subjects  
a. Study Protocol used by study personnel at Greater Gaborone DHMT Clinics:   
 
Enrolment, follow up visits and sample collection 
• All participant recruitment will occur in up to eight DHMT clinics in Greater Gaborone. Study staff will 
be trained in the study’s methods, protocol, and human subjects research, and will conduct eligibility 
screening on all patients attending their first ANC visit, following a simple, standard checklist of 
eligibility criteria. Staff will read all eligible women a brief description of the study. Interested women 
will then be read aloud the study consent form in their preferred language, which will provide specific 
information about sexually transmitted infections, the consequences and treatment of those infections, 
and study risks and benefits, and then will be invited to participate in the study. Staff will collect basic 

Title: The diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae in pregnant 
women to prevent adverse neonatal consequences.” 
 
 
 
19 Sept 2022 
15
de-identified information from clinic logs (i.e., age, gestational age, HIV status) to use for descriptive 
analysis of the general ANC patient population. 
 
• Study personnel will inform potential participants about the risks and benefits of study participation 
and answer any questions they may have concerning the study protocol. Study personnel will then 
ask interested eligible women sign an informed consent for participation (consent form in appendix). 
 
• To ensure retention, those providing informed consent will be asked to provide detailed contact 
information (e.g., phone numbers and “home address” for self, family, friend/neighbor) to ensure 
follow-up. To develop and maintain a strong relationship with participants, study staff will conduct 
welcome phone calls within 3 days of enrollment, and check in with participants during regular ANC 
clinic visits. Clinic and study staff will contact participants who do not return for scheduled ANC visits 
and encourage return for care. We will flag participant charts so that clinic staff will notify study staff 
on the date of delivery.  
 
• During the antenatal visits (1st antenatal care and a visit in the 3rd trimester), participants in the STI 
testing group will be asked to provide self-administered vaginal swab samples for CT/NG testing by 
the GeneXpert and cytokine testing by Luminex. Participants will also be asked to provide additional 
samples that will be stored for testing of Trichomonas vaginalis, bacterial vaginosis, and/or 
Mycoplasma genitalium. These samples will be stored at the BHP lab for no longer than 24 months. 
Participants will also be asked to provide a plasma sample, collected by a trained phlebotomist, for 
cytokine testing by Luminex. 
 
• During the first antenatal visit, a sub-sample of participants in the standard of care group will be asked 
to provide two self-administered vaginal swabs for testing after study completion. These samples will 
be immediately de-identified and stored at the BHP lab for no longer than 24 months.  
 
• During the 1st antenatal care visit, participants in the syphilis PoC clinic will be asked to provide a 
blood sample for either dual HIV/syphilis or rapid syphilis point-of-care testing, which is offered in 
addition to the standard of care. 
 
• During postnatal care, all participants will self-collect a vaginal sample for CT/NG testing by the 
GeneXpert. Participants in the testing group will self-collect an additional vaginal sample and will 
provide a plasma sample for cytokine analysis. Those who test positive will be offered infant testing 
via ocular/nasopharyngeal samples. Infant samples will be collected by a trained study nurse. 
Participants will also be asked to provide additional samples at the postnatal visit that will be stored 
for testing of Trichomonas vaginalis, bacterial vaginosis, and/or Mycoplasma genitalium. These 
samples will be stored at the BHP lab for no longer than 24 months. 
 
• Postnatally, a sub-set of 35 participants who were treated for CT/NG, an STI syndrome or syphilis at 
any point during the study will be asked additional questions by study staff, expanding on partner 

Title: The diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae in pregnant 
women to prevent adverse neonatal consequences.” 
 
 
 
19 Sept 2022 
16
notification and treatment experiences. These patients will additionally be asked if they are willing to 
refer their partner to participate in a semi-structured interview. If so, they will sign an informed consent 
allowing the study team to contact their partner(s). 
 
• Specimens for CT/NG and syphilis testing will be processed onsite at the antenatal clinics by study 
personnel. 
 
• Specimens for Trichomonas vaginalis, bacterial vaginosis, and/or Mycoplasma genitalium testing will 
be stored for up to 24 months at BHP.   
 
• The self-collected swabs and EDTA plasma aliquots for cytokine measurement will be stored at -80 C. 
The swabs and EDTA plasma will be transported in dry ice to Stanford University for processing. 
Luminex will be used to measure the concentrations of 48 cytokines (including IL-1a±, IL-1b≤, IL-6, IL-
7, IL-8, IL-10, granulocyte macrophage colony- stimulating factor [GM-CSF], interferon-y≥ inducible 
protein [IP]-10, monocyte chemoattractant protein [MCP]-1, MIP-1a±, MIP-1b≤, and tumor necrosis 
factor [TNF]-a±). These cytokines were selected based on previously identified associations with 
genital infection.(34) Additionally the pathogen-specific and SAC Ct results will be automatically 
generated for each self-collected vaginal swab sample tested using the Xpert CT/NG assay at the first 
postnatal visit. 
 
Results 
• Results of the CT/NG testing will be available to the women after 90 minutes, placed in the medical 
record, and communicated to medical providers. Efforts will be made to provide results to participants 
on the same day. 
 
• Results for syphilis will be available to women after 20 minutes, placed in the medical record, and 
communicated to medical providers. Efforts will be made to provide results to participants on the 
same day. 
Treatment 
• Women diagnosed with CT will receive a single dose of oral azithromycin (1 gram) and women 
diagnosed with NG will receive a single dose of oral azithromycin (1 gram) and a ceftriaxone 250 mg 
injection will be administered on site according to standard of care per Botswana MOH guidelines.  
 
• Infants testing positive will receive treatment according to Botswana guidelines (20mg/kg of 
azithromycin (liquid formulation) for CT, injection of 50mg/kg of ceftriaxone for NG) and referrals  
 
• Women testing positive for syphilis either through the dual syphilis/HIV or rapid syphilis test will be 
counseled according to Botswana guidelines, including assessing previous syphilis diagnoses and 
treatment and penicillin allergies. Treatment will also be initiated according to national guidelines. As 
Botswana is a low syphilis prevalence setting (<5%), the national guidelines follow WHO 
recommendations and currently recommend Benzathine penicillin 2.4MU IM weekly for 3 weeks with 
alternative treatments for pregnant women with penicillin allergies. For women with reactive 

Title: The diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae in pregnant 
women to prevent adverse neonatal consequences.” 
 
 
 
19 Sept 2022 
17
treponemal tests, the standard of care RPR test titre results will be provided to the participant’s 
physician to inform further treatment/management. 
Test of cure 
• A test of cure will be performed approximately four weeks after initial treatment for CT/NG during an 
antenatal care visit to document clearance of infection. If patients are positive on repeat testing, they 
will be retreated and given additional counseling regarding CT and NG and additional efforts will be 
made to follow up, counsel and treat any new sex partners or old sex partners who have not been 
treated. 
Partner Services 
• All study staff will be trained on appropriate and effective methods for counselling women on how to 
communicate test results to partner(s) (from the past 60 days) in a nonthreatening and blameless 
manner. (For example, it’s important to clarify that the duration of sexually transmitted infections 
during pregnancy is unknown, many infections are asymptomatic, and infections do not have 
implications about partner fidelity). Additionally, participants will be given several options for partner 
services: 1) No disclosure: In the case of risk for intimate partner violence (IPV). 2) Supportive 
disclosure: where the participant is assisted by trained study staff either in person or over the phone in 
notifying their partners. Also, participants will be given the option of bringing partners to the study 
clinics for counselling and treatment. 3) Expedited partner therapy: where the participant can bring 
CT/NG treatment to partners prior to examination by a healthcare provider. IPV: will be proactively 
assessed and managed for all participants, not only those testing STI positive. To that end, we will 
use a validated screening tool adapted from Rabin RF et al.[1] to assess participant risk for intimate 
violence and other negative partner responses to notification of an STI. Based on the screening 
results, when appropriate, IPV risk will be mitigated by referring women to local support services 
available in the country for free counseling and support, the NGO Botswana Family Welfare 
Association (BOFWA; http://www.bofwa.org.bw/). We will also provide a 24-hour emergency contact 
line through which participants can receive information and support and referrals in general. 
 
• Efforts will be made to ensure that partners return to the study clinics for treatment. Those who return 
will receive the stat doses of azithromycin and ceftriaxone or Benzathine penicillin as described above 
for participants. In cases where participants and study staff are concerned about partner access to 
prompt clinical evaluation and treatment for CT/NG, partners will be offered expedited therapy (EPT) 
whereby participants will be provided with treatment to bring home for partner(s).[46] For CT, EPT will 
consist of a single dose of oral azithromycin (1 gram). For NG, EPT will consist of oral cefixime 
(400mg) and a single dose of oral azithromycin (1 gram).[47] 
 
8. Research involving survey, questionnaires: There is a questionnaire for demographic data and 
medical history for participants that will be filled out by study staff and a brief semi-structured 
interview on partner notification and treatment experiences that will be conducted by study staff in a 
sub-set of participants.     
 
 
9. FDA approval (e.g. for experimental drugs, etc.)---- N/A 

Title: The diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae in pregnant 
women to prevent adverse neonatal consequences.” 
 
 
 
19 Sept 2022 
18
 
10. Data collection, Storage and Confidentiality: 
 
Data will be collected electronically using REDCap. Data stored in REDCap will not have personal 
identifiers and personal identification codes will be used. A code sheet linking the data to the 
subject’s personal identification will be stored separately from the REDCap data in a locked 
research office in the BHP offices. Only authorized study personnel will have access to the 
REDCap data and the linking form. Each staff member will have their own secret login and REDCap 
logins will be monitored by the study manager.  
 
11. Risk/Benefit Assessment (12-17)  
 
a. Potential Risks and Discomforts: 
The potential risks encountered during the study are: 1) CT and NG testing 2) Specimen collection.  3) 
Questionnaire. 4) Data analysis 
 
Conducted by Study Personnel at Princess Marina: 
The primary potential risks to participants due to study participation are psychological and social in nature. 
Physical risks from a self-collected vaginal specimen are negligible. Unlike HIV and syphilis testing,  
screening for CT or NG is not a routine part of the standard of care for pregnant women in Botswana, 
participants in the comparison group have no risk greater than that normally incurred during a typical 
antenatal care visit; i.e. mild physical or psychological discomfort. IPV is a possible risk when women test 
positive for CT/NG. IPV will be proactively assessed and managed for all participants using a validated 
screening tool to assess participant risk for intimate violence and other negative partner responses to 
notification of an STI. Based on the screening results, when appropriate, IPV risk will be mitigated by 
referring women to local support services available in the country for free counseling and support. We 
will provide a 24-hour emergency contact line through which participants can receive information and 
support and referrals in general. Also, women will be given several options for partner notification and 
treatment. 
 
A. Psychological: Participants could experience psychological distress such as anxiety when discussing 
issues related to personal experiences, sexual health, or pregnancy. However, we do not expect any 
serious events to occur based on our experience across multiple previous studies, including our pilot 
study with this same population in Botswana. Participants may experience some stress related to the 
knowledge of STI status.  Participants will be given information and education about the nature and 
consequences of all infections and treatment, and those testing positive (including newborns) will be 
provided treatment as per standard treatment protocols. The likely harmful consequences of learning 
one’s STI status are low. 
 
B. Social: Participation in this study may cause social harm (e.g., discrimination, false assumptions, 
rumors) if the participation of the participant becomes known to others. One of the more significant risks 
is notification of sexual partners about positive results of CT or NG testing, which is an important step to 
protect the health of the partners and their future contacts. It is possible that notifying partners about a 
positive CT or NG test could put the participant at risk for IPV. Given this, we will provide IPV prevention 
counseling and will take steps to mitigate and monitor such outcomes, providing intensive participant 
support as needed. 
 

Title: The diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae in pregnant 
women to prevent adverse neonatal consequences.” 
 
 
 
19 Sept 2022 
19
C. Alternative treatments and procedures: Participants from the target population invited to participate 
will be able to decline participation, with no consequence to them. 
 
b. Risk Classification:  Minimal. 
 
c. Minimizing Risks:   
Informed consent and strict confidentiality will be rigorously enforced to minimize risks to participants 
and their partners. All study personnel will receive training in the informed consent process.  
 
Additional protections will be put in place for participants under the age of 18 including reporting 
requirements for abuse and referrals to social workers and added support for partner notification. We 
have also added an adolescent health expert to our team to advise on adolescent-related issues. 
 
Trained providers will be available in case participants have difficulty in any part of the specimen 
collection such as pain or discomfort or in the case of any adverse event. 
 
Participants will be provided with a number whereby a member of the clinic staff and the Principal 
Investigator, Dr. Chelsea Morroni, may be contacted to answer questions or in case of emergency or 
any questions regarding their informed consent. 
 
d. Potential Benefits:  
 
Conducted by study personnel: 
Diagnosis and treatment of CT/NG may prevent the development of future complications in 
reproductive health as well as transmission of undiagnosed infections to newborns and sexual partners, 
and reduce the risk of adverse birth outcomes. Diagnosis and treatment of CT/NG may address 
symptoms among infants. Participants in the syphilis PoC testing clinics will have access to rapid 
syphilis results, which may increase the likelihood of treatment access for those who test positive and 
may reduce the need for non-routine follow-up visits.  
 
Potential benefits to society include determination of the benefits (in terms of infections cured and 
prevention of vertical transmission of STIs and subsequent adverse outcomes) of integration of routine 
CT/NG screening into antenatal care in Botswana. The study will also provide valuable operational data 
regarding of patient follow-up and treatment, as well as insights into the formation and revision of 
antenatal care policies, guidelines and funding levels. Results will serve to estimate prevalence of 
CT/NG infections among asymptomatic pregnant women in Gaborone, Botswana.  
 
Risk/Benefit Ratio: The benefits to the participants in diagnosis and treatment of CT/NG infection 
outweigh the risks of physical or personal discomfort from specimen collection. As syphilis testing is 
already part of the standard of care, there are no additional testing risks. As described above, 
confidentiality of study data will be rigorously maintained. The benefits of PoC syphilis testing include 
same day results and treatment. 
 
12. Therapeutic Alternatives:  Participation in the study protocol is voluntary.  Patients declining 
participation will still receive treatment from their physician, including STI screening, according to 
current standards of care. 
 
13. Financial Considerations: 

Title: The diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae in pregnant 
women to prevent adverse neonatal consequences.” 
 
 
 
19 Sept 2022 
20
 
a. Payment for Participation: Participants will receive 50 Pula to help compensate for attendance at 
each study visit including baseline, test of cure, third trimester, postnatal, and infant testing visits. 
 
b. Financial Obligations of the Subjects: There are no costs to participate in the study. 
 
14. Emergency Care and Compensation for Research-Related Injury: 
Minimal risk is associated with participation in this study.  A telephone number is provided on the 
informed consent form for participants to speak with a study clinician if they believe they have 
experienced a research-related injury. Study personnel will help participants obtain appropriate care or 
direct them to the hospital’s emergency room if they believe they have a life-threatening emergency.    
 
15. Informed Consent: 
a. Capacity to consent:  All subjects will have the capacity to give informed consent. 
 
b. Personnel Inviting Participants: 
Study personnel will invite potential participants and will help them to understand the informed consent 
document. Only after all the information has been provided and the potential participants have all their 
questions about the study answered, will staff ask them to sign the informed consent form to participate 
in the study. The process of obtaining informed consent will be finished before collection of data or 
specimens. Study personnel will give a copy of the consent document to the participants to keep. They 
will exclude any potential participant who is unable to understand the informed consent, severely 
mentally disabled, or does not meet age criteria. They will keep consent forms in a locked office in 
Gaborone.  All study staff involved in recruiting and consenting participants will have completed the 
Botswana equivalent of a Human Research Subjects certification program before the beginning of the 
study. 
 
16. Process of Consent: 
After verification of eligibility, the informed consent process is as follows: 
 
• The study interviewer says: “We would like to read through this consent form with you because we want 
to make sure that you understand everything in it and that we answer any questions you may have.”  
The study interviewer reviews the consent form, paragraph by paragraph, and stops as needed for any 
questions or clarifications. 
 
• The interviewer asks the potential participant short questions about the consent to make sure that she 
fully understands the study. This process gives the interviewer time to assess the potential participant 
to make sure there are no “gross Impairments” that would invalidate the informed consent (e.g. 
participant does not fully understand the study, is intoxicated, etc.) and make study participation 
unethical and prohibited. 
 
• If the potential participant agrees to participate in the study, the study interviewer then signs and dates 
the consent form. The participant’s study code is then written on the consent form.   
 
• The subject is given a copy of the consent form. 
 
17. Comprehension of the Information Provided: 

Title: The diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae in pregnant 
women to prevent adverse neonatal consequences.” 
 
 
 
19 Sept 2022 
21
The study personnel will ask participants to restate in their own words the study’s objectives and what 
participation involves to determine if they fully understand the informed consent. If the participant does 
not understand the protocol, the study interviewer will explain again the content of the consent form.  If 
the participant is still unable to understand the protocol after it is reviewed again, she will be excluded 
from participation in the study.  
 
18. Information withheld from Subjects:  Not applicable. 
 
19. Consent/Assent forms:  The consent form used in the study is in the appendix.  
 
20. Conflicts of Interest 
The investigators involved in this study have no conflicts of interest to report. 
 
21. Study Timeline 
 
Table 1: Study Timeline 
Start 
Finish 
Duration 
Component and Task Name 
 
 
 
I. Preparation and Piloting 
Jan 2020 
Dec 2020 
12 mo. 
II. Enrollment and Follow-up (CT/NG) 
Feb 2021 
Mar 2022 
11 mo. 
III. Enrollment (syphilis post-test group) 
August 2022 
Feb 2023 
7 mo. 
IV. Follow-up only (CT/NG) 
Apr 2022 
Dec 2022 
9 mo. 
V. Data Analysis and Dissemination 
Jan 2023 
May 2023 
5 mo. 
 
22. Dissemination Plan: 
Test results and educational information will be shared with patients. Results of the study will be 
compiled into a report and presented to the Ministry of Health. Results will also be presented at local 
and international scientific conferences. Additionally, the study will be described in an article that will be 
submitted for publication in a peer-reviewed journal and results will be presented at national and 
international meetings. Finally, the results of the study will be shared with patients and staff at 
Gaborone City Clinics through presentations and posters. 

Title: The diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae in pregnant 
women to prevent adverse neonatal consequences.” 
 
 
 
19 Sept 2022 
22
REFERENCES 
 
1. 
Rabin RF, Jennings JM, Campbell JC, Bair-Merritt MH: Intimate partner violence 
screening tools: a systematic review. American journal of preventive medicine 2009, 
36:439-445.e434. 
2. 
Hammerschlag MR RP, Gelling M, Tsumura N, Jule JE, Kutlin A,: Use of polymerase chain 
reaction for the detection of Chlamydia trachomatis in ocular and nasopharyngeal 
specimens from infants with conjunctivitis. Pediatr Infect Dis J 1997, 16:293. 
3. 
Yip PP CW, Yip KT, Que TL, Kwong NS, Ho CK,: The use of polymerase chain reaction 
assay versus conventional methods in detecting neonatal chlamydial conjunctivitis. J 
Pediatr Ophthalmol Strabismus 2008, 45:234-239. 
4. 
Centers for Disease Control and Prevention: Sexually Transmitted Diseases Treatment 
Guidelines, 2015. Accessed from: http://wwwcdcgov/std/tg2015/tg-2015-printpdf 2015. 
5. 
Faro CJ, Hollier LM, Bishop K: Comparison of vaginal cytokine collection methods. 
American journal of reproductive immunology (New York, NY : 1989) 2006, 55:315-320. 
6. 
Masson L PJ, Liebenberg LJ, et al.: Genital inflammation and the risk of HIV acquisition 
in women. . Clin Infect Dis 2015, 61:260-269. 
7. 
Tighe P NO, Todd I, et al.: Utility, reliability and reproducibility of immunoassay 
multiplex kits. Methods 2013, 61:23-29. 
8. 
Van Den Heuvel A, Smet H, Prat I, Sands A, Urassa W, Fransen K, Crucitti T: Laboratory 
evaluation of four HIV/syphilis rapid diagnostic tests. BMC infectious diseases 2019, 
19:1. 
9. 
World Health Organization: The Global Elimination of Congenital Syphilis: rational and 
strategy for action. 2007. 
10. 
Glick H, Doshi J, Sonnad S, Polsky D: Economic Evaluation in Clinical Trials, 2nd Edition. 
Oxford, United Kingdom: Oxford University Press; 2015. 
11. 
Baud D, Regan L, Greub G: Emerging role of Chlamydia and Chlamydia-like organisms 
in adverse pregnancy outcomes. Current opinion in infectious diseases 2008, 21:70-76. 
12. 
Cotch MF, Pastorek JG, 2nd, Nugent RP, Hillier SL, Gibbs RS, Martin DH, Eschenbach DA, 
Edelman R, Carey JC, Regan JA, et al: Trichomonas vaginalis associated with low birth 
weight and preterm delivery. The Vaginal Infections and Prematurity Study Group. 
Sex Transm Dis 1997, 24:353-360. 
13. 
Gencay M KM, Saikku P, Puolakkainen M, Raivio K, Koskela P, Vaheri A.: Chlamydia 
trachomatis seropositivity during pregnancy is associated with perinatal 
complications. Clinical infectious diseases : an official publication of the Infectious Diseases 
Society of America 1995, 21:424-426. 
14. 
Johnson HL GK, Zenilman JM, Erbelding EJ.: Sexually transmitted infections and adverse 
pregnancy outcomes among women attending inner city public sexually transmitted 
diseases clinics. Sexually Transmitted Diseases 2011, 38:167-171. 
15. 
Locksmith G GP: Infection, antibiotics, and preterm delivery. Semin Perinatol 2001, 
25:295-309. 
16. 
Ngassa P, Egbe J: Maternal genital Chlamydia trachomatis infection and the risk of 
preterm labor. Int J Gynaecol Obstet 1994, 47:241-246. 
17. 
Rours G, Duijts L, Moll H, Grends L, de Groot R, Jaddoe V, Hofman A, Steegers E, Mackenbach 
J, Ott A, et al: Chlamydia trachomatis infection during pregnancy associated with 

Title: The diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae in pregnant 
women to prevent adverse neonatal consequences.” 
 
 
 
19 Sept 2022 
23
preterm delivery: a population-based prospective cohort study. European journal of 
epidemiology. European Journal of Epidemiology 2011, 26:493-502. 
18. 
Silva M, Florencio G, Gabiatti J, Amaral R, Eleuterio J, Goncalves A: Perinatal morbidity 
and mortality associated with chlamydial infection: a meta-analysis study. The 
Brazilian journal of infectious diseases 2011, 15:533-539. 
19. 
Liu B RC, Clarke M, Jorm L, Hunt J, Ward J.: Chlamydia and gonorrhoea infections and 
the risk of adverse obstetric outcomes: a retrospective cohort study. Sexually 
Transmitted Infections 2013, 89:672-678. 
20. 
Wood CR: Gonococcal Infections in Neonates and Young Children. Sexually and 
Perinatally Transmitted Diseases in Childhood-Presentations, Evaluations, and Implications 
2005, 16:258-270. 
21. 
Hammerschlag MR: Chlamydial and gonococcal infections in infants and children. 
Clinical infectious diseases: an official publication of the Infectious Diseases Society of 
American Journal of Obstetrics and Gynecology 2011, 53:S99-102. 
22. 
Gomez GB, Kamb ML, Newman LM, Mark J, Broutet N, Hawkes SJ: Untreated maternal 
syphilis and adverse outcomes of pregnancy: a systematic review and meta-analysis. 
Bull World Health Organ 2013, 91:217-226. 
23. 
Schachter J GM, Sweet RL, Holt J, Jordan C, Bishop E. : Prospective study of perinatal 
transmission of Chlamydia trachomatis. JAMA: the journal of the American Medical 
Association 1986, 255:3374-3377. 
24. 
Hammerschlag M, Chandler J, Alexander E, English M, Koutsky L: Longitudinal studies on 
chlamydial infections in the first year of life. Pediatr Infect Dis J 1982, 1:395. 
25. 
Laga M, Plummer F, Nzanze H, al. e: Epidemiology of ophthalmia neonatorum in Kenya. 
Lancet 1986, 2:1145-1149. 
26. 
Mudau M, Kojima N, Peters R, Feucht U, De Vos L, Olivier D, Joseph Davey D, McIntyre J, 
Klausner J, Medina-Marino A: Postpartum prevalence and Incidence of Chlamydia 
trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis among Human 
Immunodeficiency Virus-Infected women in South Africa and the transmission of 
maternal infections to newborns. In submission 2018. 
27. 
Zar HJ, Van Dyk A, Yeats JK, Hanslo D: Chlamydia trachomatis lower respiratory tract 
infection in infants. Annals of tropical paediatrics 1999, 19:9-13. 
28. 
Adachi K N-SK, Klausner JD,: Chlamydia Trachomatis Infection in Pregnancy: The 
Global Challenge of Preventing Adverse Pregnancy and Neonatal Outcomes in Sub-
Saharan Africa and Asia. BioMed Research International 2016, eprint ahead of 
publication. 
29. 
Adachi K, Xu J, Yeganeh N, Camarca M, Morgado MG, Watts DH, Mofenson LM, Veloso VG, 
Pilotto JH, Joao E, et al: Combined evaluation of sexually transmitted infections in HIV-
infected pregnant women and infant HIV transmission. PLoS One 2018, 13:e0189851. 
30. 
Adachi K, Klausner JD, Bristow CC, Xu J, Ank B, Morgado MG, Watts DH, Weir F, Persing D, 
Mofenson LM, et al: Chlamydia and Gonorrhea in HIV-Infected Pregnant Women and 
Infant HIV Transmission. Sexually Transmitted Diseases 2015, 42:554-565. 
31. 
World Health Organization: WHO publishes new estimates on congeital syphilis. 2020. 
32. 
Newman L, Rowley J, Vander Hoorn S, Wijesooriya N, Unemo M, Low N, Stevens G, Gottlieb 
S, Kiarie J, Temmerman M: Global Estimates of the Prevalence and Incidence of Four 

Title: The diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae in pregnant 
women to prevent adverse neonatal consequences.” 
 
 
 
19 Sept 2022 
24
Curable Sexually Transmitted Infections in 2012 Based on Systematic Review and 
Global Reporting. PLOS One 2015, 10:Published online. 
33. 
Wynn A, Ramogola-Masire D, Gaolebale P, Moshashane N, Sickboy O, Duque S, Williams E, 
Doherty K, Klausner JD, Morroni C: Prevalence and treatment outcomes of routine 
Chlamydia trachomatis, Neisseria gonorrhoeae and Trichomonas vaginalis testing 
during antenatal care, Gaborone, Botswana. Sex Transm Infect 2017. 
34. 
Medline A, Joseph Davey D, Klausner JD: Lost opportunity to save newborn lives: 
variable national antenatal screening policies for Neisseria gonorrhoeae and 
Chlamydia trachomatis. International Journal of STD & AIDS 2016, 28:660-666. 
35. 
World Health Organization: Global Strategy for the Prevention and Control of Sexually 
Transmitted Infections: 2006-2015 Accessed from: 
http://wwwwhoint/hiv/pub/toolkits/stis_strategy[1]enpdf 2007. 
36. 
Mayaud P, Mabey D: Approaches to the control of sexually transmitted infections in 
developing countries: old problems and modern challenges. Sex Transm Infect 2004, 
80:174-182. 
37. 
Romoren M, Sundby J, Velauthapillai M, Rahman M, Klouman E, Hjortdahl P: Chlamydia 
and gonorrhoea in pregnant Batswana women: time to discard the syndromic 
approach? BMC Infect Dis 2007, 7:27. 
38. 
Romoren M, Velauthapillai M, Rahman M, Sundby J, Klouman E, Hjortdahl P: 
Trichomoniasis and bacterial vaginosis in pregnancy: inadequately managed with 
the syndromic approach. Bull World Health Organ 2007, 85:297-304. 
39. 
Romoren M, Rahman M, Sundby J, Hjortdahl P: Chlamydia and gonorrhoea in pregnancy: 
effectiveness of diagnosis and treatment in Botswana. Sex Transm Infect 2004, 80:395-
400. 
40. 
Silveira MF, Ghanem KG, Erbelding EJ, Burke AE, Johnson HL, Singh RH, Zenilman JM: 
Chlamydia trachomatis infection during pregnancy and the risk of preterm birth: a 
case-control study. Int J STD AIDS 2009, 20:465-469. 
41. 
Romoren M, Sundby J, Velauthapillai M, Rahman M, Klouman E, Hjortdahl P: Chlamydia 
and gonorrhoea in pregnant Batswana women: time to discard the syndromic 
approach? BMC infectious diseases 2007, 7:Published online. 
42. 
Shava E, Moyo S, Zash R, Diseko M, Dintwa EN, Mupfumi L, Mabuta J, Mayondi G, Chen JY, 
Lockman S, et al: Brief Report: High Rates of Adverse Birth Outcomes in HIV and 
Syphilis Coinfected Women in Botswana. J Acquir Immune Defic Syndr 2019, 81:e135-
e140. 
43. 
Romoren M RM: Syphilis screening in the antenatal care:  a cross-sectional study from 
Botswana. BMC Int Health Hum Rights 2006, Jul 8. 
44. 
Shannon CL, Bristow C, Hoff N, Wynn A, Nguyen M, Medina-Marino A, Cabeza J, Rimoin A, 
Klausner JD: Acceptability and Feasibility of Rapid Chlamydial, Gonococcal, and 
Trichomonal Screening and Treatment in Pregnant Women in 6 Low- to Middle-
Income Countries. Sex Transm Dis 2018, 45:673-676. 
45. 
Mudau M, Peters RP, De Vos L, Olivier DH, D JD, Mkwanazi ES, McIntyre JA, Klausner JD, 
Medina-Marino A: High prevalence of asymptomatic sexually transmitted infections 
among human immunodeficiency virus-infected pregnant women in a low-income 
South African community. Int J STD AIDS 2018, 29:324-333. 

Title: The diagnosis and treatment of Chlamydia trachomatis and Neisseria gonorrhoeae in pregnant 
women to prevent adverse neonatal consequences.” 
 
 
 
19 Sept 2022 
25
46. 
Centers for Disease Control and Prevention: Expedited Partner Therapy. Accessed from:  
https://wwwcdcgov/std/ept/ 2016. 
47. 
Hogben M, Kidd S, Burstein GR: Expedited partner therapy for sexually transmitted 
infections. Current opinion in obstetrics & gynecology 2012, 24:299-304. 
 APPENDIX 
  
Informed Consent and questionnaire are attached.