The primary outcome of this study is the diagnostic performance of the FlashDx STI multiplex assay for the detection of the 6 STIs evaluated in comparison with the CE-IVD–certified Mikrogen PCR assays serving as the laboratory reference standard.

Diagnostic performance will be assessed using a predefined analytical checklist developed for this study to systematically evaluate assay accuracy. Results obtained with the FlashDx sSPRT STI panel will be compared with the Mikrogen ampliCube STD reference PCR assays. Samples yielding concordant results across both methods will be considered resolved. In cases where results are discordant, a third independent PCR assay will be performed, and the final infection classification will be assigned based on majority agreement, whereby a target will be considered positive only when at least 2 of the 3 assays produce a positive result. Diagnostic performance of the FlashDx platform will then be evaluated against this final resolved reference outcome. Sensitivity will be calculated as the proportion of true positives among all infected cases (true positives divided by the sum of true positives and false negatives), while specificity will be calculated as the proportion of true negatives among all noninfected cases (true negatives divided by the sum of true negatives and false positives). Positive predictive value and negative predictive value will also be calculated, along with corresponding confidence intervals using standard binomial methods.

Secondary outcomes include the generation of epidemiological data on the prevalence of multiple STIs within the study population, with external laboratory validation of results. In addition to CT and NG, the multiplex panel allows detection of US, MG, MH, and TV, providing a broader overview of STI distribution among PrEP users in western Kenya.

This study will use a comparative cross-sectional design aimed at validating the performance of the FlashDx STI multiplex assay for detecting STIs and estimating the prevalences of genital CT and NG.

The study will be conducted in Kisumu, Kenya, focusing on PrEP users. Study activities will be carried out at the Kenya Medical Research Institute (KEMRI) Centre for Microbiology Research (CMR) Research Care and Training Program (RCTP) testing site located at Lumumba Sub-County Hospital. This site was selected due to its established infrastructure for HIV and STI research, its access to populations with a high burden of STIs, and its capacity to support molecular diagnostic testing using the in-house Cepheid GeneXpert.

The study enrolled 400 PrEP users aged 15 years to 55 years who may have been part of existing KEMRI PrEP cohorts or PrEP delivery points in Kisumu County. This specific cohort was selected to evaluate the effectiveness and applicability of the FlashDx STI multiplex assay in a population with a high burden of STIs who is already engaged in preventative health measures. The use of PrEP was an eligibility criterion for enrollment because individuals engaged in PrEP are considered a priority population for the prevention of curable STIs, given their high incidence, interest in longitudinal preventative services, and willingness to take pills for prevention. These participants will be retained in the study unless they choose to discontinue participation.

Eligible participants had to be willing and able to provide written informed consent and had to be confirmed as being HIV-seronegative according to the national HIV testing algorithm. Participants had to also be actively receiving PrEP at the time of enrollment. Recruitment occurred through pre-existing PrEP research cohorts at KEMRI-CMR-RCTP as well as through PrEP delivery clinics in the Kisumu area. Individuals were excluded if they had received prolonged antibiotic therapy, defined as more than a 14-day course within the month prior to enrollment, or if they had any active, clinically significant medical or psychiatric condition that, in the judgment of the site investigator or designee, could interfere with safe participation in the study.

To accurately estimate the prevalence of STIs among sexually active women in Kisumu, Kenya, we based our calculations on an assumed CT prevalence of 10%, as indicated by collated studies from Sohaili et al [39] (range: 5%‐15%). The goal was to detect this prevalence with a precision of 5% (d) at a 99% confidence level (z=2.576).

To calculate the sample size, we used the Cochran formula for sample size estimation of a proportion:

where p is the population size, e is the margin error, and z is the z value extracted from a z table.

To account for the variability in estimated prevalence (range: 5%‐15%), we performed a sensitivity analysis. For a prevalence of 5%, rounding up, the sample size would be approximately 127 participants. For a prevalence of 15%, rounding up, the sample size would be approximately 339 participants.

Based on this calculation, a minimum sample size of approximately 239 participants would be required to estimate STI prevalence with the specified precision. Sensitivity analyses using the lower and upper bounds of the expected prevalence range (5%‐15%) resulted in estimated sample sizes ranging from approximately 127 to 339 participants.

In addition, this study also aims to evaluate the diagnostic performance of the FlashDx assay relative to the reference PCR method. For diagnostic validation studies, larger sample sizes are desirable to ensure sufficient numbers of positive cases for reliable estimation of sensitivity, specificity, and agreement statistics. Assuming a CT prevalence of approximately 10%, a sample of 400 participants is expected to yield roughly 40 positive cases, which provides a reasonable basis for estimating diagnostic performance measures and conducting discrepancy analyses.

Therefore, a target sample size of 400 participants was selected to ensure adequate statistical power.

Participants were recruited using two complementary strategies. The primary approach involved a nested recruitment design in which eligible participants were invited from existing KEMRI cohorts enrolled in ongoing studies at the KEMRI-CMR-RCTP site. These participants were particularly suitable for inclusion because they already undergo routine screening for bacterial STIs such as CT and NG as part of established research protocols. Individuals who met the eligibility criteria were approached by study staff; provided with detailed study information, including a participant information sheet; and invited to provide written informed consent prior to enrollment.

If recruitment from existing KEMRI cohorts was insufficient or unavailable, participants were also recruited from PrEP delivery centers in Kisumu and surrounding areas. In this case, the experienced community outreach team at the KEMRI study site implemented community-based mobilization strategies to support recruitment. These efforts involved collaboration with community health volunteers, peer educators, youth peer providers, and other local gatekeepers to disseminate study information and encourage participation. Recruitment activities could include the distribution of printed and electronic information, education, and communication materials; engagement through social media platforms such as WhatsApp and Facebook; participation in community events for information sharing; and partnerships with local educational institutions to provide health talks and awareness sessions about the study.

Since recruitment occurred from both established research cohorts and community PrEP clinics, there is potential for selection differences between these groups. To address this, baseline demographic, behavioral, and clinical characteristics will be compared by recruitment source to assess potential selection bias.

Throughout the recruitment process, strict attention was given to protecting participant confidentiality and minimizing the risk of stigma. Potential participants were not approached individually in public or group settings, and recruitment messages were carefully reviewed to ensure cultural appropriateness and community acceptability. Individuals who declined participation or who did not meet the eligibility criteria were, where appropriate, provided with information about alternative studies or referred to standard HIV prevention and STI services available in the community.

Samples were tested within Kenya where feasible. The samples were also shipped to the Netherlands for further testing and validation of results. The testing procedures are described in the following sections.

Participants self-collected urine samples to test for STIs. Urine samples were chosen because they are suitable for STI testing using the FlashDx STI multiplex assay, the Cepheid GeneXpert, and the CE-IVD-D Mikrogen assay. The FlashDx system requires 120 μL of urine to run. The urine specimen collection occurred as follows. The study assistant labeled the urine collection container with the participant identification number and the date of sample collection before providing it to the participant. The participant details were then captured in the laboratory request form.

To obtain good specimans, participants were advised how to produce and collect a satisfactory urine sample in a cup using the following instructions: The cup is very clean. Do not open it until you are ready to use it. Provide first-catch urine (20-50 mL) in a urine collection cup free of any preservatives. Screw the cap on tightly so it does not leak. Put the cup into the box or bag that the study staff provided. Give the cup containing the urine sample to the staff.

The study staff stored the cup containing the urine sample in the cooler box containing ice packs ready for use in the laboratory.

All STI screening was completed with POC testing in Kisumu, Kenya, using the FlashDx STI panel. The urine samples were also sent to the Netherlands to be tested with the reference CE-IVD-D Mikrogen assay. These diagnostics are described in detail in the following sections.

Urine samples collected during the study were aliquoted and stored at −80 °C at the KEMRI research laboratory. Samples were subsequently exported to the Netherlands for confirmatory testing under a material transfer agreement and in accordance with both Kenyan and Dutch regulatory requirements governing biological sample transport and research use [40].

The CE-IVD Mikrogen PCR assays are designed for various pathogen detections and are certified under the In-Vitro Diagnostics Directive 98/79/EC. These assays are known for their high sensitivity and specificity, providing reliable diagnostic results. These assays are compatible with most common real-time PCR cycler and various RNA/DNA extraction methods. The technology is CE-IVD certified.

For this study, the ampliCube STD panel 1 is used for real-time PCR detection of CT, NG, and MG. Moreover, the ampliCube STD Panel 2.1 is used for the detection of TV, MH, Ureaplasma parvum, and Ureaplasma urealyticum (Figure 1).

The diagnostic results obtained using the Mikrogen assay will in turn be compared with the results obtained using the FlashDX RUO tests. The presence of the different STIs will then be analyzed for alignment or discrepancy.

Participants will attend the study site for 1 primary visit. This visit will be a screening visit where potential participants will provide informed consent and be assessed against inclusion and exclusion criteria. Once participants are confirmed as eligible, questionnaires will be administered to collect demographic and behavioral information. Following this, a 4-mL urine sample will be collected from each participant. Of this sample, 120 µL will be allocated for STI screening using the FlashDx Assay. If necessary, an additional 1 mL will be used for testing with the CEPHEID GeneXpert assay. The remaining sample will be stored and shipped to the Netherlands for further testing with the Mikrogen assay.

Participants may return to the study clinic for interim visits for any reason and will undergo STI testing and treatment if symptoms are present. As participants are part of PreP cohorts and may be a part of concurrent KEMRI trials, they will also undergo routine testing for bacterial STIs such as CT and NG.

Laboratory testing will be conducted by qualified staff, and STI diagnoses will be reviewed by the study team. All participants diagnosed with an STI will receive same-day treatment or be asked to return to the research clinic for treatment using WHO and Kenya standard therapies. A follow-up visit will be scheduled 2 weeks after completing STI treatment to conduct a test to determine if the infection was cured, ensuring the effectiveness of the treatment and resolution of the infection.

The FlashDx testing platform was integrated into the KEMRI Kisumu setting, with a trained researcher from the Netherlands assisting in its implementation and providing training to local staff on sample handling and testing procedures. The researcher transferred his knowledge of the system to local staff. Additionally, the study results will be shared with local policymakers and decision-makers in the field.

Participants diagnosed with CT or NG through routine clinical testing using the GeneXpert CT/NG system were contacted by a member of the study team and invited to return to the study clinic for treatment at a time convenient for them. Treatment is provided free of charge and follows Kenyan national and WHO guidelines [41]. Uncomplicated chlamydia infections are treated with a single oral dose of azithromycin, while gonorrhea infections are treated with a single intramuscular dose of ceftriaxone combined with a single oral dose of azithromycin. Participants with suspected complicated infections, such as pelvic inflammatory disease, receive extended treatment with ceftriaxone followed by a 2-week course of azithromycin and metronidazole as clinically indicated.

Participants treated for a bacterial STI were asked to return to the clinic approximately 2 weeks after treatment for a test-of-cure visit and encouraged to inform their partners to seek testing and treatment. Participants developing STI symptoms during the study may return for further evaluation and treatment. All treatments follow Kenyan Ministry of Health and WHO standard-of-care guidelines and are provided through the study clinic or affiliated health facilities.

Self-collected urine samples are tested to collect data on STI organism-specific diagnosis.

Participants completed a research assistant-administered demographic, sexual, and behavioral questionnaire to collect the data shown in Table 3. The questionnaire was adapted from validated instruments used in previous KEMRI studies and hence in line with WHO recommendations.

The study team will monitor the progress and safety of the study through regular internal review and reporting. Monthly summary reports will track participant accrual, recruitment sources, and key baseline characteristics. In addition, quarterly reports will summarize pooled study data, including data completeness, specimen collection rates, and any reported adverse events. Participant safety will be reviewed on an ongoing basis, with particular attention to the relationship between any reported adverse events and study procedures. Monthly monitoring will also include reviews of premature study discontinuations, treatment discontinuations, and the reasons for withdrawal to ensure protocol adherence and study integrity.

Participants may be withdrawn from the study under several circumstances. These include a participant’s request to withdraw from the study at any time; determination by the investigator that the participant is at significant risk of noncompliance with the study protocol or that continued participation may pose potential harm; or a decision by oversight bodies such as the institutional review board (IRB), ethics committee, the study funder, or the Office for Human Research Protections.

Participants may feel embarrassed or worried about STI and HIV testing conducted during the study. Anxiety or worry may also from providing urine samples, though the participants will do this in a self-collected way. Study questionnaires may make participants uncomfortable or embarrassed when discussing sexual behavior or partners. All study staff are trained to provide professional care in clinical sample collection and counseling. Counseling is available to participants who need it based on their study experiences.

Additional considerations apply to participants who are minors (aged 15‐17 years). Adolescents may face greater concerns related to privacy, stigma, or disclosure of sexual health information than adult participants. To minimize these risks, study procedures are conducted in private settings, and staff use age-appropriate communication to ensure that participants understand the study procedures and their rights. Confidentiality protections are strictly maintained in accordance with IRB-approved protocols and Kenyan ethical guidelines governing research involving adolescents. Counseling services are available to provide support and guidance for minors who may experience anxiety or emotional distress related to sexual health discussions or testing.

Study staff strive to protect participants’ privacy and confidentiality. However, participants’ involvement in the study could become known to others, potentially resulting in social harms such as unfair treatment, discrimination, or problems with family and community acceptance. Participants might face issues with their partners or experience intimate partner violence. Counseling and referrals are available if needed.

Participants receive free STI testing and treatment, which is not readily available in public clinics. Prompt treatment of STIs, including asymptomatic infections, can reduce the risk of pelvic pain, infertility, and other complications that untreated STIs can cause. Participants also receive clinical assessment and counseling related to sexual and reproductive health.

Minor participants (aged 15-17 years) may particularly benefit from access to confidential sexual health services, health education, and counseling provided by trained health care professionals. These services may help adolescents better understand STI prevention, safe sexual practices, and available health care resources while also facilitating early diagnosis and treatment where needed.

Participants are reimbursed for travel costs and compensated for time spent during study visits, in accordance with local guidelines and regulatory approvals. Each participant will receive 500 Kenyan shillings (US $3.86) for the study.