We are conducting rapid HIV, CT, NG, syphilis, and HCV testing among adolescents aged 15-24 years at community-based organizations in Los Angeles and New Orleans. Our partner community-based organizations cater to GBTY, homeless youth, youth with a history of mental health disorders, and youth with a history of incarceration. Study participants receive STI testing at 4-month intervals for 24 months, totaling 1 baseline visit and 6 follow-up visits. When a participant receives a positive STI test result, they are linked to care at a nearby medical clinic or provided with antibiotic treatment by the interviewing staff. We hypothesize that by providing rapid STI testing among high-risk adolescent populations, we will find STI prevalences higher than the national averages for adolescents. We intend to evaluate the acceptability of testing as the uptake of screening among eligible participants, and we intend to evaluate the feasibility of treatment as the proportion of participants who test positive for an STI and receive treatment. We expect that by providing same-day testing results and treatment, we will be able to provide quicker time to treatment and higher treatment rates than with traditional lab-based testing. We will compare our time to treatment and treatment rates with historical data from AIDS Healthcare Foundation, Los Angeles. We also intend to evaluate other treatment outcomes such as cure rates, reinfection rates, and partner treatment rates. For objectives of other components of the ATN CARES study, refer to other ATN CARES protocol papers [20-24]. For specific power analyses, refer to the ATN CARES paper by Swendeman et al [22].

The Institutional Review Board of the University of California, Los Angeles has approved the study protocol (16-001674-AM-00006). We will report any protocol deviations or indications of adverse events to the Institutional Review Board. The study was registered on ClinicalTrials.gov (NCT03134833) on April 28, 2017.

We selected rapid STI tests according to performance, availability, and cost. Table 1 shows the sensitivity and specificity values of each test.

HIV antigen and antibody screening are done using the Determine HIV-1/2 Ag/Ab Combo test (Alere Inc) (Figure 2) [30]. This test is a point-of-care lateral flow strip that detects both HIV-1 and HIV-2 antibodies and the HIV-1 p24 antigen using 50 µL of fingerstick whole blood. The window period is 12-26 days, and results are ready in 20-40 minutes. The test is Clinical Laboratory Improvement Amendments (CLIA) waived and Food and Drug Administration (FDA) approved [25,31].

We will perform HIV RNA and DNA screening with the Xpert HIV-1 Qual test (Cepheid) (Figure 3) [32]. The test is a point-of-care qualitative in vitro HIV test, detecting HIV-1 RNA and DNA. The HIV-1 Qual test requires 100 µL of whole blood, and results are available in 90 minutes [26]. The test is approved for use in the European Union and undergoing the approval process with the FDA. Our study is the first in the United States to use the test, and results are available as research use only.

The HIV-1 Qual test can detect HIV infection an average of 5 days earlier than a p24 antigen test. Therefore, this test is done to detect acute HIV infections that we may not be able to detect with the Alere HIV test.

Syphilis screening is done using the Syphilis Health Check, a rapid point-of-care treponemal antibody test (Diagnostics Direct) (Figure 4) [33]. The test uses 50 µL of whole blood, and results are available in 10 minutes. The Syphilis Health Check is the only FDA-approved rapid syphilis test [27].

We will perform HCV screening with the HCV Rapid Antibody Test (OraSure Technologies), a rapid point-of-care assay used for the detection of HCV antibodies (Figure 5) [34]. The test uses whole blood and gives results in 20-40 minutes. The test has a waiver from the CLIA and is approved by the FDA [28].

Finally, we will perform CT and NG screening using the Xpert CT/NG Assay (Cepheid) (Figure 3). The test is a qualitative in vitro real-time polymerase chain reaction test for the detection of CT and NG. Results are available in 90 minutes [29]. The test is FDA approved for urine samples and vaginal swabs. However, it is also verified in accordance with CLIA for pharyngeal and rectal swabs [35]. Male participants self-collect pharyngeal and rectal swabs as well as a urine sample, while female participants self-collect pharyngeal, rectal, and vaginal swabs.

Interviewing staff conducts all STI and HIV rapid testing at community-based recruitment sites. Interviewers are typically Bachelor of Arts-level paraprofessionals with little prior experience related to rapid diagnostic testing. Some have previously received phlebotomy training, but most receive phlebotomy training upon hiring. Interviewers receive training and certification in state-specific HIV counselor training. HIV counselor training includes training on fingerpricking, conducting different types of rapid HIV tests, interpreting results, and providing counseling regarding safe sex practices. We also coordinate specific training in Los Angeles and New Orleans for each diagnostic test. The respective diagnostic test companies (Alere, Cepheid, Diagnostics Direct, and OraSure) conduct the training. We evaluate interviewers on their ability to properly collect fingerprick blood and on their ability to correctly interpret test results. We conduct repeat diagnostic test training every 6 months to ensure interviewers continue to correctly perform tests. A binder with step-by-step test instructions is at every site in case any questions arise.

We also train interviewers on how to instruct participants to self-collect rectal swabs, pharyngeal swabs, vaginal swabs, and urine samples. For rectal swabs, we provide an image to show the acceptable level of fecal contamination on the swab (Figure 6).

Interviewers use Fleshlite (Austin) models to demonstrate how to self-collect vaginal and rectal swabs (Figures 7 and 8) [36], while they use a mirror to locate the tonsils and demonstrate how to self-collect a pharyngeal swab.

Finally, we train interviewers on how to administer treatment for CT and NG infections. A physician prescribes the antibiotics, and interviewers are trained by the physician on how to properly deliver antibiotic therapy. Training includes information about antibiotic mechanisms, pharmacokinetics, potential adverse effects, partner therapy, retesting, and STI counseling. Interviewers practice providing treatment using sample scenarios to demonstrate competence.

While we perform HIV, CT, NG, and syphilis testing at every recruitment site, we only perform HCV testing at sites with populations at higher risk of HCV (history of incarceration or drug use). Every study participant receives every STI test unless they specifically choose to opt out. Opting out does not affect eligibility or reimbursement.

At baseline, the rapid HIV test is done as part of the eligibility screening to determine if the participant is HIV-infected or -uninfected. We determine eligibility based on a risk assessment, with a minimum risk score necessary for enrollment. Inclusion criteria and risk scoring are explained in detail in the ATN CARES protocol papers by Rotheram-Borus et al and Comulada et al [21,24]. If a participant is eligible for the study, they are enrolled and receive the additional STI testing. An interviewer with phlebotomy certification draws blood for use in the HIV RNA/DNA test, the syphilis test, and the HCV test at certain sites. The participant self-collects their urine sample or pharyngeal, rectal, and vaginal swabs. Clients are encouraged to stay until their test results are available.

Routine follow-up appointments occur at 4-month intervals for 2 years. However, if a patient reports potential STI exposure or STI symptoms to the interviewer, they will be invited for testing at any point during the study.

Participants receive their test results on the same day as the testing. Whenever possible, they are told the results in person. If the participant needs to leave their appointment before results are available, the interviewer asks the participant how they would like to receive the results and then communicates the results with them through a phone call, text, or email. When a patient receives a positive test result, they are either referred to a partner medical clinic or to their primary care provider to receive treatment, or they are provided with treatment by the interviewing staff. All partner medical clinics agreed to and signed an STI treatment protocol that is in accordance with Centers for Disease Control and Prevention recommendations.

If the participant elects to seek treatment at a clinic, the interviewer works with the participant to find a clinic that is geographically convenient, and the study organizes free transport to the clinic via the Uber app. Interviewers counsel participants on the importance of partner treatment and safe sex practices. They also follow up with study participants to ensure they were able to receive treatment, and study staff obtains records of the treatment from the clinic.

For syphilis infections, we will always refer the participant to a clinical provider since follow-up blood work and a penicillin injection may be required. For CT and NG infections, it is up to the participant if they prefer to be referred to a clinic or receive same-day antibiotic treatment from the interviewing staff. The interviewers treat vaginal, urethral, and pharyngeal CT infections with 1 g oral azithromycin [1]. They treat rectal CT infections with 100 mg oral doxycycline twice daily for 7 days, as evidence shows doxycycline is more effective than azithromycin in treating rectal CT [37-40]. NG infection, while often clinically treated with 1 g oral azithromycin and an injection of 250 mg ceftriaxone, is instead treated with 1 g oral azithromycin and 400 mg oral cefixime [1]. The treatment of concurrent CT and NG infections are according to the type of CT infection. If there is a vaginal, urethral, or pharyngeal CT infection in addition to an NG infection, treatment is 1 g oral azithromycin and 400 mg oral cefixime. If there is a rectal CT infection in addition to an NG infection, treatment is 100 mg oral doxycycline twice daily for 7 days and 400 mg oral cefixime. All treatment regimens follow Centers for Disease Control and Prevention guidelines and are effective treatment methods.

We prepackage treatment packs that include antibiotic instructions, antibiotics, physician contact information, water, and a snack. Every recruitment site has these treatment packs available. We also offer participants with a positive CT or NG result up to 10 expedited partner therapy packets according to the number of partners reported in the past 90 days [41]. We provide the expedited partner therapy packs according to the type of infection participants test positive for as well as the type of sex they report having with their partner. For example, if a participant tests positive for urethral CT and reports insertive anal sex, we will provide doxycycline in the expedited partner therapy pack.

The study team monitors STI prevalence to ensure that it falls within the expected range. A research assistant performs monthly quality control testing at every testing site to confirm that all tests are functioning properly. Monthly quality control testing involves running a positive and negative control sample for each rapid STI test at each site. This ensures that the tests correctly identify both positive and negative results. In addition to every month, we perform quality control testing whenever a new interviewer is conducting the tests, a new test lot number is received, or if the storage temperature falls outside the recommended range.

Interviewing staff record STI lab results on a paper lab form as well as through CommCare, a mobile data collection platform created by Dimagi (Cambridge). We then obtain the documentation of STI treatment from the medical clinics.

Using these data, we will evaluate STI prevalence, risk factors, and HIV seroconversion rates throughout the study period. We will also evaluate successful linkage to care and treatment of positive STI cases.

At the time of manuscript submission, we are in the process of making one change to our study protocol. Due to the high prevalence of a history of syphilis in our study population and the low specificity of the Syphilis Health Check, a participant with a positive Syphilis Health Check result requires additional laboratory testing. Therefore, we will obtain rapid plasma reagin titers and Treponema pallidum particle agglutination testing when a participant has a reactive Syphilis Health Check result. Quest Diagnostics will perform the rapid plasma reagin and Treponema pallidum particle agglutination tests. We anticipate that this change will significantly improve our ability to properly diagnose syphilis infections.