Healthy Expectancy Through Routine Antenatal STI Screening

Aim 1 Procedures

i. Screening, recruitment and enrolment

Investigators will identify and enroll 2000 pregnant women presenting for antenatal care (ANC) services at up to ten clinics in Gaborone and surrounding villages. All pregnant women at their first ANC visit will be screened for eligibility after standard HIV and syphilis screening per Botswana National Guidelines. Interested women who meet the age, pregnancy, gestational age, ANC visit, and residence criteria will be read a brief study description. Following confirmation of eligibility by clinic midwife (no cervicovaginitis requiring syndromic treatment), women will be provided with more detailed study information and invited to provide informed consent. Interested women will be read the study consent form, which will include information on Chlamydia trachomatis (CT) and Neisseria gonorrhoea (NG) infections, study risks and benefits, and each potential group assignment. Staff will record reasons for ineligibility or refusal and collect basic de-identified information from clinic logs to use for descriptive analysis.

Written informed consent will be obtained from each study participant prior to enrollment. Women who are unable to read and/or write will be required to have an impartial witness present during the above procedures. Participants will be offered copies of the informed consent forms. Forms will be available in English and Setswana. For eligible participants aged 15-17, parental consent will be sought from their parent/guardian (verbal consent by telephone if parent/guardian not present and documented by the study team).

All participant recruitment will occur in government clinics in Botswana. The study will be managed by a Study Coordinator and a Nurse Coordinator who will be based at study clinics, supervising a field team consisting of experienced research assistants and a qualified research nurse who are responsible for day-to-day conduct of study procedures including recruitment, enrollment and follow-up. A study clinician will also be on call for clinical questions from the study team and to dispense treatment. The Principal Investigator is responsible for the overall conduct and management of the study.

During periods of recruitment, study posters, flyers and other materials may be placed at the clinics alerting potentially eligible pregnant women about the study. All recruitment and materials will be reviewed and approved by the IRB. Study staff will be trained in the study's methods, protocol, and ethical human subjects' research.

ii. Randomization procedures

Randomization will occur following all routine antenatal procedures and enrollment into the study. Randomization procedures were informed by community engagement. Participants will be randomized in a 1:1 ratio stratified by recruitment site to ensure balanced allocation across study arms within each site. The randomization sequence will be generated and overseen by the study statistician. Randomization and allocation will be implemented through the REDCap randomization module, which will automatically assign participants to study arms after eligibility confirmation, ensuring allocation concealment throughout enrollment. Once assigned, the randomization allocation will be unblinded. To minimize influence of the unblinded nature of the study on outcomes, ongoing data monitoring will not include information about endpoints disaggregated by site or arm. Only the study statistician will review data on endpoints by arm or site.

iii. Baseline (first ANC) visit procedures

All participants: All participants will receive the standard-of care i.e., routine ANC services conducted by the clinic midwives as per the standard-of-care in Botswana, including a clinical assessment of obstetric history, health status (e.g. weight, blood pressure, anemia, urinalysis), internal pelvic exam, routine labs (e.g. complete blood count, blood type, and Rh factor), provider-initiated counseling and screening for HIV and syphilis, treatment for syphilis if required, tetanus toxoid immunization, and iron and folate supplementation. Women who test positive for HIV antibodies receive CD4 T cell and HIV-1 viral load screening and referral for specialist review and initiation of antiretroviral therapy. These procedures will be done prior to enrollment in the study. Thereafter all participants will be asked to respond to a questionnaire.

Questionnaire: An interviewer-administered questionnaire will be administered to all participants in their preferred language (e.g., English or Setswana). The questionnaire will be adapted from measures used in our previous STI screening studies or documented in literature. Data will also be extracted from the participant's obstetric record card and clinic records.

The main measures will include 1) demographics and socio-economic status; 2) obstetric, gynecological, prior medical, and sexual health history; 3) sex partner and relationship dynamics, including risk of intimate partner violence; and 4) patient direct and indirect costs associated with transportation, co-pays, potential lost wages due to waiting or scheduling additional visits. Physical, and pelvic examination findings, as well as syphilis and HIV test results from routine antenatal procedures, will be recorded.

Intervention arm: In addition to the standard-of-care described above, participants in the intervention arm will receive screening for CT and NG at their first ANC. Specimens collected for screening will include two self-collected vaginal swabs. The first swab will be used for CT/NG screening (with CT/NG assay). The second swab will be stored for future screening of STIs and vaginal infections. Results arising from future testing will not be returned to participants, as test results may no longer reflect current clinical status and therefore may not be clinically applicable.

CT/NG-positive participants will be treated using US CDC recommended treatments and will receive azithromycin 1g by mouth for CT and ceftriaxone 500 mg by injection for NG. (Note: the Maduo Study achieved a 100% cure rate among participants treated for CT and/or NG infection). Participants who test positive will receive counselling on STIs, the importance of partner notification and treatment, and abstinence from sexual intercourse until seven days after treatment. Participants who test positive will also be scheduled for a test-of-cure approximately four weeks after treatment.

All participant-facing study staff will be trained on partner(s) notification counseling. For example, it is important to clarify that the duration of CT infection during pregnancy is unknown, many infections are asymptomatic, and infections do not implicate partner fidelity. Participants will be given several options for partner services: (1) no disclosure, in the case of risk for intimate partner violence; (2) supportive disclosure, where the participant is assisted by trained study staff either in person or over the phone in notifying their partners, and given the option of bringing partners to the study clinics for counselling and treatment; and (3) expedited partner therapy, where the participant can bring treatment to partners prior to partner examination by a healthcare provider.

Standard of care arm: At the baseline and third trimester visits, the study team will ask to collect remnant urine from the routinely collected specimens. The urine specimens will be stored and used to test for CT and NG after delivery to retrospectively assess whether baseline STI prevalence is comparable between study arms and to support secondary analyses of infection dynamics during pregnancy. Because testing of stored specimens will occur retrospectively, the results may not reflect participants' current infection status at the time results become available. Individual results from retrospective testing of stored specimens will not be returned as diagnostic results. However, participants in the SOC group whose stored specimens test positive for CT and/or NG will be notified and offered CT/NG testing, with treatment provided if the repeat test is positive. In addition, all participants in the SOC will be provided with study contact information and informed that participants may request STI screening after delivery at any time while the study is ongoing.

iv. Ultrasound

After enrollment, all participants in both study arms will be booked in for an ultrasound performed by a study practitioner trained in collaboration with the University of Botswana Obstetrics and Gynaecology specialty training programme. After this, participants will be encouraged to attend all regularly scheduled ultrasounds offered through routine care.

v. Third trimester visit

The third trimester visit will take place between 28 and 32 weeks' gestation. The study team will try our best to schedule the visit to coincide with a routine ANC visit. Data collection at this visit will include information on pregnancy progress, routine monitoring and clinical examination findings, any incident HIV diagnosis and treatment initiation, healthcare visits or admissions, and medication or supplement use since enrollment. If participants had an early delivery before the visit, this information will be deduced during a routine follow-up phone call to the participant. Research nurses will be adequately trained to collect relevant perinatal history, ascertain overall well-being of the mother and newborn if it was a live birth, and link her to relevant health services if necessary.

Participants in the intervention arm will provide two self-collected vaginal swabs. The first swab will be used for CT/NG screening. The second swab will be stored for future screening of STIs and vaginal infections. Results arising from future testing will not be returned to participants, as participants may no longer reflect current clinical status and therefore may not be clinically applicable. Results, treatment, partner services and test-of-cure will be delivered as described at baseline.

vi. Data collection post-birth

Investigators will conduct a full medical record review to collect date of birth, birthweight, delivery method, spontaneous preterm birth, induced preterm labor, and C-section. The primary outcome, preterm birth, (\<37 weeks gestation), will be calculated based on delivery date, last menstrual period, and ultrasound dating. If last menstrual period and ultrasound are inconsistent, the study team will re-date based on the ultrasound alone. Investigators will also seek to determine whether the preterm birth was medically indicated (e.g. presence of preeclampsia, placental abruption, intrauterine growth restriction, fetal distress). Secondary outcomes will include infant outcomes: low birth weight (\< 2500g), and a composite measure of preterm or low birth weight; very preterm birth (\<32 weeks gestation); and preterm birth sub-types (spontaneous, medically indicated), which will be abstracted from the infant record; and pregnancy/maternal outcomes: gestational hypertension (one-two elevations of systolic (≥140) or diastolic blood pressure (≥90) after 20 weeks pregnancy), preeclampsia (hypertension with substantial proteinuria after 20 weeks pregnancy),50 and incident HIV infection will be abstracted from the obstetric record. If data are missing, investigators will check the Integrated Patient Management System (IPMS), a nationwide electronic system that include laboratory records, and thereafter follow-up with participants by phone.

v. Retention strategy To ensure high participant retention, investigators will leverage successful strategies from our prior work in Botswana. These include deploying trained and closely supervised staff, providing reasonable reimbursements to cover time and travel expenses, and collecting multiple contact methods (text, WhatsApp, phone calls) for participants and their trusted associates.

For follow-up purposes, study staff will collect partici

• Infant Outcomes: low birth weight, and a composite measure of preterm or low birth weight; very preterm birth; and preterm birth sub-types. [Time frame: Number of weeks gestation at birth, calculated from delivery date and date of last menstrual period and first trimester ultrasound dating.]
• Pregnancy/maternal outcomes: gestational hypertension, preeclampsia, incident HIV infection. [Time frame: First trimester ANC through post-delivery obstetrical follow-up]