A combined formulation of oral tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) is a component of first-line regimens for antiretroviral therapy (ART) in HIV-infected individuals.1 Tenofovir disoproxil fumarate/emtricitabine (TDF/FTC) can also be used for pre-exposure prophylaxis (PrEP).2–4 Daily oral TDF/FTC was approved by the United States (US) Food and Drug Administration (FDA) for PrEP in individuals at high risk of HIV acquisition in 2012,5 and is now recommended by the US Centers for Disease Control (CDC) and World Health Organization (WHO) for prevention of HIV infection in diverse risk groups.6,7 The IPERGAY8 study demonstrated that an event-driven nondaily TDF/FTC regimen can also reduce the risk of HIV infection in men who have sex with men (MSM).
A meta-analysis of randomized controlled trials and observational studies showed that high adherence was significantly associated with a protective effect of TDF and TDF/FTC PrEP.4 In the iPrEx study, participants in the TDF/FTC arm who had detectable levels of study drugs had a >92% risk reduction (95% CI: 40% to 99%) compared with those without detectable drugs.9 In other trials, such as Fem-PrEP10 and VOICE,11 low PrEP efficacy was attributed to low adherence to the study regimens.
HIV drug resistance can arise in individuals who become infected while using PrEP. However, most studies show that resistance to tenofovir (TFV) and FTC arises infrequently in the setting of TDF/FTC PrEP use.4,12 In a report that included results from 8 randomized clinical trials, resistance emerged in 5.9% of 305 seroconverters.3 Higher rates of TDF/FTC resistance were observed in those who had undiagnosed acute HIV infection at the time of PrEP initiation.4,12 M184I/V drug resistance mutations, which confer resistance to FTC, are seen more commonly in the setting of PrEP than the K65R mutation, which confers resistance to TDF.12 Emergence of resistance in individuals who become infected while using PrEP may limit options for subsequent ART.
Detailed characterization of seroconversion events in PrEP trials can help distinguish between infections due to infrequent dosing/nonadherence and true breakthrough infections, and can provide information on the relationship between PrEP exposure and emergence of drug resistance. In this study, we characterized incident HIV infections in the HIV Prevention Trials Network (HPTN) 067/ADAPT trial.13–15 The trial included a once-weekly directly observed therapy (DOT) phase followed by a period of self-administered therapy (SAT) that included 3 study arms with different TDF/FTC PrEP regimens (daily, time-driven, and event-driven). The trial was designed to compare the coverage of sexual events, number of doses needed for coverage, and self-reported side-effects/symptoms associated with daily vs. nondaily PrEP use. The DOT phase of the HPTN 067/ADAPT trial was implemented before randomization to establish individual pharmacokinetic (PK) parameters to help interpret PK-based adherence assessments performed during the SAT phase of the study. Unfortunately, a priori estimates of the half-life of TFV diphosphate (TFV-DP) in peripheral blood mononuclear cells (PBMCs) were too long, and weekly DOT dosing was too infrequent to achieve consistently measurable drug concentrations 1 week after dosing. Although inclusion of the DOT phase did not provide the desired information (PK parameters in individual study participants), it did provide an opportunity to evaluate infections that occurred in the setting of an infrequent (once-weekly) observed drug dosing. Infections that occurred during the SAT phase of the trial were also analyzed. Data analyzed in this report include self-reported PrEP use and results obtained with a panel of HIV diagnostic tests, HIV viral load testing, antiretroviral (ARV) drug testing, and HIV drug resistance testing.
Samples and data were obtained from the HPTN 067/ADAPT study, a phase 2 randomized, open-label trial of the use of oral TDF/FTC (300 mg TDF/200 mg FTC tablet) PrEP among HIV-uninfected individuals (NCT: 01327651, 2011–2014). The study enrolled 622 participants in Cape Town, South Africa (women who have sex with men, N = 191), Bangkok, Thailand (MSM and transgender women, N = 193), and New York, USA (MSM and transgender women, N = 238). The study included a 6-week lead-in period with 5 once-weekly directly observed TDF/FTC doses (DOT phase; doses were administered at enrollment and weeks 1–4 with no dosing at week 5). At the 6-week visit, participants were randomized to one of 3 PrEP regimens: daily (once daily dosing); time-driven (twice weekly dosing with an additional dose after sexual intercourse); and event-driven (24–48 hours before and within 2 hours after sexual intercourse). The study drug was dispensed and participants were tested for HIV infection at monthly study visits during the SAT phase (weeks 6–30). The final study visit was at week 34. All participants were instructed not to take more than 2 pills per 24-hour period, or more than 7 pills per week.
After randomization, weekly interviews were conducted by phone or in person with an interviewer who was not involved in other study activities. Data from electronic dose monitoring (Wisepill) were discussed to determine which device opening events were reflective of dosing and which were not; the date and time of sex events were also recorded.
HIV testing was performed at enrollment, at weeks 4 and 6, and at monthly follow-up visits. Two HIV rapid tests were performed in parallel at study sites; tests used included the Uni-gold Recombigen HIV test (Trinity Biotech PLC, Bray, County Wicklow, Ireland); the Determine HIV-1/2 test (Abbott Laboratories, Abbott Park, IL); and the OraQuick Advance Rapid HIV-1/2 Antibody Test (Orasure Technologies Inc., Bethlehem, PA). PrEP was discontinued if one or both of the HIV rapid tests were reactive. In these cases, HIV infection was confirmed at study sites using a qualitative HIV RNA assay (APTIMA HIV-1 RNA Qualitative Assay; Hologic Gen-Probe INC., San Diego, CA). Additional HIV testing was performed retrospectively at the HPTN Laboratory Center (Johns Hopkins University, Baltimore, MD) using a panel of assays (Fig. 1).
Drug Resistance Testing
Drug resistance testing was performed retrospectively at the HPTN Laboratory Center for plasma samples with viral loads >400 copies/mL. Two methods were used for drug resistance testing: the ViroSeq HIV-1 Genotyping System (Abbott Molecular, Des Plaines, IL) and next generation sequencing (NGS). HIV subtyping was performed using HIV pol sequences obtained from the ViroSeq system, as described.16 NGS was performed using viral RNA that was extracted from plasma samples using the ViroSeq system; methods used for NGS are described in Supplemental Digital Content 1, http://links.lww.com/QAI/A997. HIV drug resistance reports were generated using the Stanford University HIV drug resistance database.17
ARV Drug Testing
ARV drug testing was performed retrospectively by the HPTN Laboratory Center by liquid chromatography tandem mass spectrometry using plasma samples (all 3 sites), PBMC samples (South Africa and Thailand), and dried blood spots (DBS) samples (US).18–20 The lower limit of quantification for these assays are as follows: plasma: TFV and FTC 0.31 ng/mL,18 PBMC: TFV-DP 2.5 fmol/sample, FTC triphosphate (FTC-TP) 0.1 pmol/sample, the average lower limit of quantification based on cells assayed per sample is 0.57 fmol/106 cells for TFV-DP and 0.014 pmol/106 for FTC-TP,20 DBS: TFV-DP 31.25 fmol/punch, FTC-TP 0.125 pmol/punch.21 Plasma and PBMC drug concentrations were interpreted based on results from a dose ranging PK study with directly observed TDF/FTC dosing.20
All study participants provided written informed consent for participation in the HPTN 067/ADAPT study. The study was approved by the participating academic institutions and ethics committees for each study site.
Twelve study participants acquired HIV infection (8/191 in South Africa; 2/193 in Thailand; 2/238 in the US, Table 1). HIV subtypes were consistent with subtypes prevalent at each study site (South Africa: subtype C; Thailand: CRF01_AE; US: subtype B). All 12 participants received once-weekly observed TDF/FTC doses in the DOT phase. Six participants were not randomized because of HIV infection or pregnancy (Fig. 1A), and 6 were randomized at the 6-week study visit (2 in each study arm; Figs. 1B–D).
Detection of HIV Infection Using Third Generation HIV Rapid Tests
Participants were tested with 2 third generation HIV rapid tests at each study visit. Retrospective testing using fourth generation HIV tests, HIV RNA tests, and other assays revealed that the rapid tests often missed HIV infection. In 9/12 cases, both of the rapid tests were nonreactive at the first HIV positive visit. In these cases, HIV-infected participants continued to use PrEP until their infection was detected at the study site. A qualitative HIV RNA assay was positive in all 12 cases at the first HIV-positive visit; the HIV viral load was ≤400 in 4 cases. In 8/12 cases, retrospective testing revealed that participants had acute HIV infection at the first HIV-positive visit; in 5 of these cases, positive tests results were obtained for HIV RNA assays only; in the other 3 cases, one or both of the fourth generation tests were also reactive. In 2 other cases where HIV infection was missed by one or both of the rapid tests, other HIV tests indicated the presence of anti-HIV antibodies. In one case, the Western blot was indeterminate, and the discriminatory assay was negative (case 4); in the other case, the Western blot was indeterminate, and the discriminatory test was positive (case 5). In 4/12 cases, one or both of the rapid tests were still nonreactive at the seroconversion visit (cases 1, 2, 5, and 7); in one case (case 7), both rapid tests were nonreactive at multiple study visits.
Analysis of HIV Infection in Participants Who Were Not Randomized to a PrEP Regimen
Six participants were not randomized (Fig. 1A); these participants received only once-weekly TDF/FTC dosing in the DOT phase of the trial. Three had acute HIV infection at enrollment that was not detected at the study sites (cases 1–3), and 2 acquired HIV infection during the DOT phase (cases 4 and 5). HIV infection was diagnosed at the study site at the 4-week visit in 4 cases; those participants did not receive TDF/FTC at week 4. In the fifth case (case 4), HIV infection was diagnosed at the 5-week visit; that participant received all 5 DOT doses. One participant was not randomized to a PrEP regimen because of pregnancy (case 6); this participant acquired HIV infection between the 22- and 26-week study visits.
ARV drug testing was performed using plasma samples collected at week 4 (before dosing) and week 5. TFV and/or FTC was detected in plasma at one or both visits in 5 of the 6 cases; FTC was detected at week 6 in one of 2 participants who received TDF/FTC at week 4 (case 6). PBMC testing was performed for the 5 participants. TFV-DP and/or FTC-TP was detected at week 4, 5, or 6 in all 5 cases. Plasma testing was performed at weeks 22 and 26 for the participant who was not randomized because of pregnancy (case 6); TFV and FTC were not detected at those visits.
Analysis of HIV Infection in Participants Who Were Randomized to a PrEP Regimen
The remaining 6 participants acquired HIV infection after randomization (Figs. 1B–D). PrEP was discontinued when one or both of the HIV rapid tests were reactive. Two participants were randomized to the daily PrEP study arm (Fig. 1B). In case 7, a female participant had undiagnosed acute infection at the 6-week randomization visit; HIV testing was negative at week 5, indicating that this participant was infected 1–2 weeks after receiving 5 once-weekly DOT doses. This participant continued to use PrEP for 3–4 months after infection (from week 6 to week 22), until the infection was detected by HIV rapid testing at the study site; the participant reported taking 84.4% of the assigned doses during this period. Study drugs were detected in plasma and PBMCs at all visits tested during the SAT phase (weeks 10–22), but the concentration of drugs detected at some visits were consistent with less than daily PrEP use (TFV concentration corresponded to 7 doses/wk at week 10, ≥4 doses/wk at week 14, 7 doses/wk at week 18, and 7 doses/wk at week 22). At the visit before the participant was diagnosed with HIV infection at the study site (week 18), the concentration of TFV-DP in PBMC was consistent with 1 dose/wk (based on comparison to her data from the DOT phase); the discrepancy between plasma and PBMC drug concentrations at week 18 suggests that this participant may have taken the drug shortly before the study visit (“white coat effect”). HIV viral loads were persistently low in this case (<400 copies/mL at all but one visit; 650 copies/mL at week 18). Fourth generation HIV assays were reactive after infection, but had low signal-to-cutoff ratios (<2.5 for the ARCHITECT assay; <9 for the Bio-Rad assay). The APTIMA qualitative HIV RNA assay was initially positive, but was negative at subsequent visits; this assay has a limit of detection of 40 copies/mL HIV RNA. Resistance testing results were not obtained in this case because the viral load was low at all visits after HIV infection. In case 8, the participant acquired HIV infection between weeks 10 and 14 (4–8 weeks after randomization). Study drugs were detected in only 1 plasma sample and 1 DBS sample collected during the SAT phase. These results suggest that the participant was not adherent to the daily PrEP regimen.
Two participants were randomized to the time-driven study arm (Fig. 1C). In case 9, the participant was infected between weeks 18 and 22 (12–16 weeks after randomization). Study drugs were detected in only 2 of 4 plasma samples collected during the SAT phase (weeks 10–22); low levels of study drugs were detected in PBMC samples at 2 of these visits. These results suggest that the participant was not adherent to the time-driven study regimen. In case 10, HIV infection was diagnosed at the study site at week 18. Retrospective testing revealed that the participant had acute HIV infection at the previous visit (week 14, 8 weeks after randomization); PrEP was continued in the 4-week interval between these 2 visits. Study drugs were detected in plasma and PBMCs at all visits during the SAT phase (weeks 10–18); the drug concentrations were higher than expected for the time-driven regimen (TFV concentration corresponded to 7 doses/wk at week 10, multiple doses/day at week 14, and 7 doses/wk at week 18). The concentration of TFV-DP in PBMC at week 18 was also higher than expected (consistent with 7 doses/wk). Self-reported data indicated that this participant took a dose every 3–4 days for the first 6 weeks after randomization. She continued to take 2 pills/wk over the next 2 weeks, but took those doses at irregular intervals. She reported taking a pill 4 days before the acute infection visit, after taking no pills for 7 days.
Two participants were randomized to the event-driven study arm (Fig. 1D). In case 11, HIV infection was diagnosed at the study site at week 34, 4 weeks after the end of the SAT phase. Retrospective testing revealed that the participant had acute HIV infection at the previous visit (week 30, 24 weeks after randomization). Study drugs were detected in only 1 of 7 plasma samples and 2 of 3 PBMC samples collected during the SAT phase (at weeks 10–30). In case 12, HIV infection was diagnosed at the study site week 22. Retrospective testing revealed that the participant had acute HIV infection at the previous visit (week 18, 12 weeks after randomization); PrEP was continued in the 4-week interval between these 2 visits. Study drugs were detected in only 2 of 4 plasma samples and 1 of 2 PBMC samples collected during the SAT phase (at weeks 10–22), indicating infrequent PrEP use. Both participants reported infrequent sex events with low adherence to the event-driven regimen (42% of assigned doses taken in case 11; 67% of assigned doses taken in case 12).
HIV Drug Resistance
Samples from the 12 seroconverters were tested for HIV drug resistance. Mutations associated with resistance to the study drugs were detected in 3 cases using NGS. This included 2 cases where participants had acute HIV infection at enrollment (case 1: K65R was detected in 24.7% of sequences; case 2: M184I was detected in 3.5% of sequences), and one case where the participant was randomized to the time-driven arm (case 10: K65R was detected in 3.9% of sequences; M184I was detected in 62.3% of sequences). In the first 2 cases (cases 1 and 2), participants received only 4 once-weekly DOT doses of PrEP (at study enrollment and at weeks 1–3) before resistance was detected. Using a genotyping assay based on population sequencing (ViroSeq), resistance to study drugs was detected in only 1 of the 3 cases (case 10); in that case, only 1 of the 2 drug resistance mutations was detected (M184I). Resistance to other ARV drugs was detected in 2 cases [resistance to nonnucleoside reverse transcriptase inhibitors (cases 2 and 8)].
This report presents analysis of 12 cases of HIV infection in the HPTN 067/ADAPT trial. This included 3 cases where participants were acutely infected at enrollment, 2 cases where participants were infected during the once-weekly DOT phase, and 6 cases where participants were infected after randomization to one of 3 self-administered PrEP regimens. In the last case, a participant was not randomized because of pregnancy and was infected during the follow-up period. In the 7 of 8 cases that occurred in the context of PrEP use, participants had infrequent PrEP dosing (eg, once-weekly DOT only) or low/suboptimal adherence. In one case, adherence was high during most of the follow-up period; infection followed a 7-day period with no dosing (one missed dose in the time-driven arm).
The findings in this report highlight the higher diagnostic yield of sensitive assays for HIV diagnosis in the setting of PrEP use. In 9/12 cases, HIV infection was missed by 2 third generation HIV rapid tests at the first HIV-positive visit; in 8 of these cases, rapid tests were nonreactive at the first HIV-positive visit because the participant had acute HIV infection. Frequent detection of acute HIV infection in this cohort likely reflected the short intervals between study visits. In 7 cases, one or both of the fourth generation assays were also nonreactive at the first HIV-positive visit. In 3 cases, one or both of the rapid tests also missed infection at subsequent study visits, where both fourth generation tests were reactive. Failure to detect HIV infection using third generation rapid tests resulted in continued PrEP use in 8 cases. In one case, PrEP use was continued for 3–4 months after infection. In 3 of these cases, participants developed resistance to the study drugs (see below). In the iPrEx study, most HIV-infected individuals who had nonreactive HIV rapid tests had a positive HIV RNA test.22 In HPTN 067/ADAPT, 4 of 8 participants who had acute infection at the first HIV-positive visit had a very low viral load (≤400 copies/mL); in these cases, infection was only detected using a sensitive, FDA-cleared qualitative HIV RNA assay. In the case where PrEP use was continued for 3–4 months after HIV infection, HIV RNA levels dropped below the level of detection for this sensitive assay, and the signal-to-cutoff ratios for both fourth generation tests remained low. This case illustrates that prolonged daily PrEP use in some individuals with undiagnosed HIV infection may be associated with low-level antibody production and sustained viral suppression.
In a study in rhesus macaques, PrEP was associated with delayed antibody maturation and low viral load.23 In the Partners PrEP study, TDF or TDF/FTC PrEP was associated with delayed anti-HIV antibody formation (delayed time to develop a positive Western blot).24 In the CAPRISA study, antibody maturation was delayed in women receiving vaginal TFV gel for PrEP.25 In that study, antibody maturation was evaluated using assays developed for cross-sectional HIV incidence estimation.25 In contrast, detection of HIV seroconversion was not delayed in 2 other PrEP trials.9,26 These findings suggest that it may be difficult to diagnose HIV infection in some individuals who are taking TDF-based PrEP, particularly if less sensitive assays are used for HIV screening, and that PrEP use in cohorts and populations could also impact estimation of HIV incidence using cross-sectional surveys.27
In HPTN 067/ADAPT, resistance to study drugs was detected in 3 of 12 seroconverters. In 2 cases, resistance mutations were detected by NGS only; in the third case, one mutation was detected by routine HIV genotyping (M184I), and one was detected by NGS only (K65R). This demonstrates the additional diagnostic yield in this setting when more sensitive methods are used for analysis of HIV drug resistance. In 2 of these cases, participants with undiagnosed acute HIV infection were only exposed to 4 once-weekly doses of TDF/FTC; this indicates that even very limited exposure to PrEP is sufficient to induce resistance in individuals with early/acute HIV infection.
Detailed characterization of seroconversion events in this study revealed that all 12 incident infections occurred in the setting of infrequent PrEP dosing or low/suboptimal adherence, and that drug resistance can arise with minimal exposure to PrEP. This report also highlights the importance of using sensitive assays for HIV diagnosis and resistance testing in the setting of PrEP. Identification of HIV infections before starting PrEP and prompt discontinuation of PrEP in those who become infected after starting PrEP should reduce the risk of HIV drug resistance.
The authors thank the HPTN 067 study team, study participants, and laboratory staff at study sites and the HPTN Laboratory Center for their contributions to the study. The authors also thank Dr. Timothy Holtz and Dr. Anupong Chitwarakorn for their support of the HPTN 067 study at the site in Thailand. We would also like to acknowledge the lifelong contributions of Mr. Patrick Flaherty who passed away this year.
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