Protocol and Study Population

The protocol was approved by public health authorities and by ethics committees in France (Comité de Protection des Personnes Ile de France IV) and Canada (Comité d’Ethique de la Recherche de Montreal). All participants provided written informed consent. Full details with respect to the study design can be found in the study protocol, available with the full text of this article at NEJM.org.

Inclusion criteria were HIV-negative status, an age of at least 18 years, and male or transgender female sex among participants who have sex with men and who are at high risk for HIV infection (defined as a history of unprotected anal sex with at least two partners during the past 6 months). Exclusion criteria included positive results on testing for hepatitis B surface antigen, chronic infection with hepatitis C virus, a creatinine clearance of less than 60 ml per minute (as assessed by means of the Cockroft–Gault equation), an alanine aminotransferase level of more than 2.5 times the upper limit of the normal range, and glycosuria or proteinuria of more than 1+ on urine dipstick testing.

Randomization and Study Procedures

Randomization was performed by means of a fixed-size block of 4 and stratified according to country. At enrollment, eligible HIV-negative participants were assigned in a 1:1 ratio to receive either either TDF-FTC or placebo. The use of a placebo was deemed to be justified because of the inconsistent efficacy of preexposure prophylaxis in previous trials and the moderate efficacy of preexposure prophylaxis in the iPrex trial among men who have sex with men.

TDF-FTC was given as a fixed-dose combination of 300 mg of TDF and 200 mg of FTC per pill. Participants were instructed to take a loading dose of two pills of TDF-FTC or placebo with food 2 to 24 hours before sex, followed by a third pill 24 hours after the first drug intake and a fourth pill 24 hours later. In case of multiple consecutive episodes of sexual intercourse, participants were instructed to take one pill per day until the last sexual intercourse and then to take the two postexposure pills. When resuming preexposure prophylaxis, participants were instructed to take a loading dose of two pills unless the last drug intake was less than 1 week earlier, in which case they were instructed to take only one pill.

Study visits were scheduled 4 and 8 weeks after enrollment and every 8 weeks thereafter. Each visit included drug dispensation with enough pills to cover the daily use of TDF-FTC or placebo between visits, pill count and adherence counseling, serum testing for HIV-1 and HIV-2, and biochemical analyses. Before each visit, participants were asked to complete at home a computer-assisted structured interview to collect information about sociodemographic characteristics, use of alcohol and recreational drugs, sexual behavior, and adherence to preexposure prophylaxis during their most recent sexual intercourse.

Standard Prevention Interventions

At every scheduled visit, participants were offered a comprehensive package of prevention services, including patient-centered, interactive counseling according to the RESPECT risk-reduction model performed by a peer community member, free condoms and gel, and diagnosis and treatment of sexually transmitted infections.15 Peer counselors were also available between visits to address participants’ needs and reinforce adherence to study medications. Vaccination against hepatitis A and B was offered to all participants who were at risk for these infections. At enrollment and every 6 months thereafter, participants were screened for syphilis (on serologic analysis) and for chlamydia and gonorrhea (by means of a specific polymerase-chain-reaction [PCR] assay performed on anal and throat swabs and urine samples). Treatment of incident sexually transmitted infections was provided according to the protocol recommendations. Postexposure prophylaxis was readily available at study sites in case of unprotected exposure to a possibly HIV-infected partner.

Primary End Point

The primary end point was the diagnosis of HIV-1 infection, which was defined as the first evidence of HIV antibodies or p24 antigen in serum with the use of a fourth-generation enzyme-linked immunosorbent assay (ELISA) for HIV-1 and HIV-2 combined or HIV-1 RNA in plasma on PCR assay. At most of the sites, investigators used the Architect HIV Ag/Ab Combo assay (Abbott) for ELISA and the RealTime HIV-1 assay (Abbott) or Cobas TaqMan HIV-1 Test, version 2.0 ( Roche), for HIV RNA PCR. Genotypic testing for drug resistance was performed on the sample obtained at the time of diagnosis to detect major resistance mutations at positions 184, 65, and 70 of the reverse transcriptase gene.16

Analysis of Adherence

Pill count was the first measure of adherence. Participants were asked to return their study-drug bottles at each visit, and a pill count of unused medication was performed. We also measured drug levels in plasma in the first participants who were enrolled. Plasma was tested for the presence of tenofovir and FTC with the use of a validated liquid chromatography–tandem mass spectrometry method with a limit of detection of 0.1 ng per milliliter for tenofovir and 0.4 ng per milliliter for FTC. This plasma assay was able to detect drugs up to 9 days after intake.17

Adherence to the study regimen during the most recent sexual intercourse was also assessed by means of computer-assisted structured interviews that were completed before each visit. Three categories of adherence were defined: correct use of preexposure prophylaxis (at least one pill taken within 24 hours before sex and one pill taken within 24 hours after sex), no use of preexposure prophylaxis (no pills taken within 48 hours before and after sex), and suboptimal use of preexposure prophylaxis (i.e., any other use).

Safety

All participants who received at least one dose of TDF-FTC or placebo were included in the safety analyses. Adverse events were recorded at each visit, regardless of the perceived association with the medication. Toxicity was graded according to the scale of the severity of adverse events in adults used by the France Recherche Nord et Sud Sida-HIV et Hépatites (National Agency of Research on AIDS and Viral Hepatitis [ANRS]).18

Study Oversight

The conduct of the trial at each study site was monitored by the Service Commun 10–Unité de Service 19 (a clinical trial center) of INSERM. Gilead Sciences donated the study medications and provided funding for the pharmacokinetics analysis but had no role in data collection, data analysis, or manuscript preparation. All the authors vouch for the completeness and accuracy of the data reported and adherence to the study protocol.

Statistical Analysis

We calculated that 64 HIV-1 seroconversion events would provide a power of 80% to detect a 50% relative reduction in the incidence of HIV-1 infection in the TDF-FTC group, as compared with the placebo group, at a two-sided alpha level of 0.05. The expected incidence of HIV-1 infection in the placebo group was 3 cases per 100 person-years. We determined that a sample size of 1900 participants would be required to achieve the target number of study end points, with 12 to 36 months of follow-up for each participant and a rate of loss to follow-up of 15 per 100 person-years.

We used a modified intention-to-treat approach for the primary analysis, in that we excluded data only from participants who were found to have HIV-1 infection before receiving the first dose of study medication or who were lost to follow-up or withdrew consent between randomization and enrollment and did not receive study medication. All participants who underwent randomization were included in an intention-to-treat analysis.

We used the Kaplan–Meier method to estimate the cumulative probability of HIV-1 infection per group and used the log-rank test to perform between-group comparisons. To assess sexual behavior over time in the two study groups, probit mixed models and binomial mixed models were used.

The study data were reviewed every 6 months by an independent data and safety monitoring board. On October 23, 2014, just after the early discontinuation of another trial of preexposure prophylaxis involving men who have sex with men, called the Preexposure Option for Reducing HIV in the UK: An Open-Label Randomization to Immediate or Deferred Daily Truvada for HIV-Negative Gay Men (PROUD) in the United Kingdom,19 the data and safety monitoring board asked for a first unblinded interim analysis of the data and subsequently recommended that the placebo group be discontinued and that all the study participants be offered on-demand preexposure prophylaxis. The present analysis includes data collected during the double-blind phase of the study up to January 27, 2015. The study is now ongoing with an open-label design.

All analyses were conducted with the use of Stata/SE software, version 12.1 (StataCorp), and SAS software, version 9.2 (SAS Institute). All P values and confidence intervals are two-sided.