Dried blood spots were collected at each monthly study visit to characterize drug adherence by measuring intracellular tenofovir-diphosphate (TFV-DP) for long-term drug exposure (~7 days). The 196 participants who had monthly DBSs available were stratified by site, gender, and the 3 patterns previously assigned for the TFV extracellular analysis (2×2×3 = 12 strata). Then 60 participants were selected, 5 from each of the 12 strata, to balance by site, gender, and the above 3 patterns were maintained. In turn, the monthly DBSs indicated by the assigned pattern were analyzed. These 60 participants contributed a total of 237 monthly DBSs for the TFV-DP intracellular analysis. The observed TFV-DP levels in our study population were categorized as follows (units of drug in fmol/mL): 0 doses per week (<912); 1 dose taken per week (≥912 and <1824); 2 doses taken per week (≥1824 and <2688); 3 doses taken per week (≥2688 and <3600); 4 doses taken per week (≥3600 and <4464); 5 to 7 doses taken

Study visits were scheduled every month until study completion. Participants were instructed to return to the clinic for evaluation in event of illness. Participants reported any adverse effects (AEs) at monthly or interim visits and were determined as serious adverse events (SAE) when at least possibly related to study drug. DAIDS Table for Grading Severity of Adult Adverse Experiences for Vaccine & Prevention Research Programs was used for grading. Definitions: Grade 3-'probably related'-strong temporal relationship to study product that cannot be explained by participant's clinical state or other factors and a causal relationship is biologically plausible. Grade 4-'definitely related'-distinct temporal relationship to administration of the study product that cannot be explained by the participant's clinical state or other factors or AE occurs on re-challenge or the AE is a known reaction to the product or chemical group or can be predicted by the product's pharmacology.

Number of sex partners was assessed at baseline and each scheduled monthly visit, for up to 12 months.

Responses to the following question refers to the number of partners reported in the past 30 days:

"In the past 30 days, with how many partners have you had sexual intercourse?"

Among 229 participants, 102 participants were women and 127 participants were men. Analyses were stratified by sex.

Number of sex acts by condom usage was assessed at baseline and each scheduled monthly visit, for up to 12 months.

Responses to the following question refers to the number of sex acts with up to 3 partners.

"In the past 30 days, how many times did you have sex with ['this partner']? When I ask about the number of times you had sex, please count each sexual act. For example, if you had 2 rounds of sexual intercourse with your partner on a single evening, count that as two times you had sex. Please remember that this only refers to vaginal and anal sex. It does not refer to oral sex."

To assess condom use by sex act, the following question was asked to assess the number of sex acts with condoms and without condoms with up to 3 partners:

"Of the ___ sex acts, how many times did you not use condoms the entire time?"

Among 229 participants, 102 participants were women and 127 participants were men. Analyses were stratified by sex.

Dried blood spots were collected at each monthly study visit to characterize drug adherence by measuring extracellular tenofovir (TFV) for recent drug exposure (~24 hours). Of 229 participants, 196 participants had monthly DBSs available for analysis. A sampling algorithm was designed to make inference to TFV and TFV-DP levels at all 12 months. For the TFV extracellular analysis, participants were randomly assigned to one of three sampling schedules, with equal probability: (a) months 1, 2, 5, 8, and 11; (b) months 1, 3, 6, 9, and 12; and (c) months 1, 4, 7, 10, and 12. These 196 participants contributed a total of 777 monthly DBSs for the TFV extracellular analysis.

Extracellular TFV detectability was defined as having a mean TFV level (of up to four measurements) equal to or greater than 5 ng/mL.

Of 229 participants, 196 participants had monthly DBSs available for analysis over a period of 12 months. These 196 participants contributed a total of 777 monthly DBSs for the TFV extracellular analysis.

Dried blood spots were collected at each monthly study visit to characterize drug adherence by measuring intracellular tenofovir-diphosphate (TFV-DP) for long-term drug exposure (~7 days). The 196 participants who had monthly DBSs available were stratified by site, gender, and the 3 patterns previously assigned for the TFV extracellular analysis (2×2×3 = 12 strata). Then 60 participants were selected, 5 from each of the 12 strata, to balance by site, gender, and the above 3 patterns were maintained. In turn, the monthly DBSs indicated by the assigned pattern were analyzed. These 60 participants contributed a total of 237 monthly DBSs for the TFV-DP intracellular analysis. The observed TFV-DP levels in our study population were categorized as follows (units of drug in fmol/mL): 0 doses per week (<912); 1 dose taken per week (≥912 and <1824); 2 doses taken per week (≥1824 and <2688); 3 doses taken per week (≥2688 and <3600); 4 doses taken per week (≥3600 and <4464); 5 to 7 doses taken

Of the 196 participants with monthly DBSs available over a period of 12 months, 60 participants were selected for TFV-DP intracellular analysis and contributed a total of 237 monthly DBSs for the TFV-DP intracellular analysis.

Study visits were scheduled every month until completion of the study and during monthly study visits, HIV testing was performed, for up to 12 months. During monthly visits, routine HIV testing was performed with two HIV rapid tests. If HIV-infection was suspected, HIV antigen-antibody (Ag/Ab) combination enzyme immunoassay (EIA) (Bio-Rad, GS HIV Combo Ag/Ab EIA) testing was performed, and RNA viral load was measured.

Study visits were scheduled every month until study completion. Participants were instructed to return to the clinic for evaluation in event of illness. Participants reported any adverse effects (AEs) at monthly or interim visits and were determined as serious adverse events (SAE) when at least possibly related to study drug. DAIDS Table for Grading Severity of Adult Adverse Experiences for Vaccine & Prevention Research Programs was used for grading. Definitions: Grade 3-'probably related'-strong temporal relationship to study product that cannot be explained by participant's clinical state or other factors and a causal relationship is biologically plausible. Grade 4-'definitely related'-distinct temporal relationship to administration of the study product that cannot be explained by the participant's clinical state or other factors or AE occurs on re-challenge or the AE is a known reaction to the product or chemical group or can be predicted by the product's pharmacology.