Pre-exposure Prophylaxis Use by Breastfeeding HIV-Uninfected Women: A Prospective Short-Term Study of Antiretroviral Excretion in Breast Milk and Infant Absorption

Background As pre-exposure prophylaxis (PrEP) becomes more widely used in heterosexual populations, an important consideration is its safety in infants who are breastfed by women taking PrEP. We investigated whether tenofovir and emtricitabine are excreted into breast milk and then absorbed by the breastfeeding infant in clinically significant concentrations when used as PrEP by lactating women. Methods and Findings We conducted a prospective short-term, open-label study of daily oral emtricitabine–tenofovir disoproxil fumarate PrEP among 50 HIV-uninfected breastfeeding African mother–infant pairs between 1–24 wk postpartum (ClinicalTrials.gov Identifier: NCT02776748). The primary goal was to quantify the steady-state concentrations of tenofovir and emtricitabine in infant plasma ingested via breastfeeding. PrEP was administered to women through daily directly observed therapy (DOT) for ten consecutive days and then discontinued thereafter. Non-fasting peak and trough samples of maternal plasma and breast milk were obtained at drug concentration steady states on days 7 and 10, and a single infant plasma sample was obtained on day 7. Peak blood and breast milk samples were obtained 1–2 h after the maternal DOT PrEP dose, while maternal trough samples were obtained at the end of the PrEP dosing interval (i.e., 23 to 24 h) after maternal DOT PrEP dose. Tenofovir and emtricitabine concentrations were quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays. Of the 50 mother–infant pairs enrolled, 48% were ≤12 wk and 52% were 13–24 wk postpartum, and median maternal age was 25 y (interquartile range [IQR] 22–28). During study follow-up, the median (IQR) daily reported frequency of infant breastfeeding was 15 times (12 to 18) overall, 16 (14 to 19) for the ≤12 weeks, and 14 (12 to 17) for the 13–24 wk infant age groups. Overall, median (IQR) time-averaged peak concentrations in breast milk were 3.2 ng/mL (2.3 to 4.7) for tenofovir and 212.5 ng/mL (140.0 to 405.0) for emtricitabine. Similarly, median (IQR) time-averaged trough concentrations in breast milk were 3.3 ng/mL (2.3 to 4.4) for tenofovir and 183.0 ng/mL (113.0 to 250.0) for emtricitabine, reflecting trough-to-peak breast milk concentration ratios of 1.0 for tenofovir and 0.8 for emtricitabine, respectively. In infant plasma, tenofovir was unquantifiable in 46/49 samples (94%), but emtricitabine was detectable in 47/49 (96%) (median [IQR] concentration: 13.2 ng/mL [9.3 to 16.7]). The estimated equivalent doses an infant would ingest daily from breastfeeding were 0.47 μg/kg (IQR 0.35 to 0.71) for tenofovir and 31.9 μg/kg (IQR 21.0 to 60.8) for emtricitabine, translating into a <0.01% and 0.5% relative dose when compared to the 6 mg/kg dose that is proposed for therapeutic treatment of infant HIV infection and for prevention of infant postnatal HIV infection; a dose that has not shown safety concerns. No serious adverse effects were recorded during study follow-up. The key study limitation was that only a single infant sample was collected to minimize venipunctures for the children. However, maternal daily DOT and specimen collection at drug concentration steady state provided an adequate approach to address the key research question. Importantly, there was minimal variation in breast milk concentrations of tenofovir and emtricitabine (respective median trough-to-peak concentration ratio ~1), demonstrating that infants were exposed to consistent drug dosing via breast milk. Conclusion In this short-term study of daily directly observed oral PrEP in HIV-uninfected breastfeeding women, the estimated infant doses from breast milk and resultant infant plasma concentrations for tenofovir and emtricitabine were 12,500 and >200-fold lower than the respective proposed infant therapeutic doses, and tenofovir was not detected in 94% of infant plasma samples. These data suggest that PrEP can be safely used during breastfeeding with minimal infant drug exposure. Trial Registration ClinicalTrials.gov, Identifier: NCT02776748


To quantify the steady state concentrations of tenofovir (TFV) and emtricitabine (FTC) in infant plasma after FTC/TDF PrEP exposure through maternal breast milk.
Decisions about the safety of breast feeding during maternal ingestion of drugs require knowledge of the amount of drug which might be present in breast milk. Understanding the concentration of FTC/TDF infants are exposed to and subsequently absorbed via breast milk when taken as PrEP by lactating women is needed to permit a thorough evaluation of the benefits and risks of maternal FTC/TDF PrEP use during lactation. We will measure infant plasma, maternal plasma, and breast milk FTC and TFV concentrations in an open-label, repeat dose study of daily FTC/TDF PrEP among HIV-1-uninfected lactating mother-infant pairs.
The primary outcomes of this study will be the steady state infant plasma FTC and TFV levels, infant plasma to maternal breast milk FTC and TFV concentration ratios, and maternal breast-milk to plasma FTC and TFV concentration ratio. A single infant plasma sample will be collected after the maternal 7 th PrEP dose. Four maternal plasma and breast-milk samples each will be collected, for trough and peak levels immediately before administration of the 7 th and 10 th dose and 1-2 hours after the 7 th and 10 th dose, respectively, two samples at each visit. More than 2.5 million persons are infected with HIV-1 annually, the majority in sub-Saharan Africa, the region with the highest prevalence. 9 In sub-Saharan Africa, the majority of new HIV-1 infections occur in women of childbearing age. 9 Pregnancy and postpartum breastfeeding represent periods of heightened HIV-1 risk for women and for their infant if acute HIV-1 infection occurs. 10 HIV-1 uninfected women who desire pregnancy with a partner who is HIV-1 infected or at high risk of being infected require protective strategies for themselves and their future children. Novel, effective HIV-1 prevention strategies remain urgently needed, particularly those that are deliverable to and useable by high-risk populations. Daily oral pre-exposure prophylaxis (PrEP) for uninfected persons to prevent HIV-1 acquisition 1, 3, 4 , peri-coitally dosed tenofovir gel 11 , and antiretroviral treatment (ART) to reduce the infectiousness of HIV-1 infected persons 12 are the most promising new approaches for decreasing HIV-1 spread. These antiretroviral-based strategies provide an HIV-1 prevention option for women who desire conception or pregnancy and that is under their control; other available HIV-1 prevention strategies of condoms and abstinence, do not allow for conception.

PrEP protects against heterosexual transmission of HIV-1
Pre-exposure prophylaxis (PrEP) with tenofovir disoproxil fumarate (TDF) alone or in combination with emtricitabine (FTC/TDF) reduces the risk of HIV acquisition in high-risk HIV-uninfected adults in a diverse geographical and at risk populations. Clinical trials have demonstrated protective efficacies of 44-75% in randomized comparisons and ~90% in case-control analyses of participants with high PrEP adherence. [1][2][3][4] In the Partners PrEP Study, a phase III, placebo-controlled trial of oral TDF and combination FTC/TDF PrEP among HIV-1 uninfected partners in 4747 HIV-1 serodiscordant in Kenya and Uganda, compared to placebo, FTC/TDF and TDF reduced risk HIV-1 acquisition in both men and women by 75% and 67%, respectively ( Table 1). The rate of serious medical events was similar across the study arms. Further evidence for the efficacy of PrEP in the Partners PrEP Study is provided by a case-cohort analysis. For subjects on the active PrEP arms who acquired HIV-1 after randomization, 31% had tenofovir detected in a plasma sample at the seroconversion visit compared with 82% of a randomly-selected samples from a subset of subjects who did not acquire HIV-1, verifying overall high adherence in the trial and demonstrating that seroconverters had low adherence (Table 2). Importantly, having detectable tenofovir in plasma was associated with a relative risk reduction for acquiring HIV-1 of 86% (TDF, p<0.001) and 90% (FTC/TDF, p=0.002)further emphasizing high protection against HIV-1 for those who were PrEP-adherent The Partners PrEP Study results reinforce findings of other recent PrEP studies: in CAPRISA 004 peri-coital tenofovir vaginal gel reduced HIV-1 risk by 39% (95% CI 6-60%, p=0.017) among 889 South African women 11 ; in iPrEx, daily oral FTC/TDF reduced HIV-1 risk by 44% (95% CI 15-63%, p=0.005) among 2499 men who have sex with men, 3 and oral FTC/TDF reduced HIV-1 risk by  4 ; in Bangkok tenofovir study, daily TDF reduced HIV-1 risk by 49% (95% CI 9·6-72·2; p=0.01) among 2413 HIV-1 uninfected adult injection drug users. 2 Across PrEP studies, adherence has been a key predictor of efficacy; largely explaining the lack of HIV-1 protection found among African women in the FEM-PrEP (using FTC/TDF) 13 and VOICE trials (using TDF, FTC/TDF, and tenofovir gel) in which <30% had detectable tenofovir in plasma. 14 Pregnancy and postpartum breast feeding are periods of heightened HIV-1 risk and women in reproductive years are a target population for PrEP to prevent HIV-1 acquisition Pregnancy and postpartum period represent periods of heightened HIV-1 risk for women and for their infant if acute HIV-1 infection occurs. Published observational data from sub-Saharan Africa indicate incidence rates during pregnancy and postpartum period as high as 16.8 and 4.7 per 100 person-years in some settings 8,[15][16][17][18] , respectively (Table 3). Gray et al using data from a cohort of HIV-1 serodiscordant couples in Rakai, Uganda and Mugo et al using data from HIV-1 uninfected women from 7 countries in east and Southern Africa both estimated a 2-fold higher risk of HIV-1 acquisition during pregnancy than non-pregnancy periods 8,17 . In a systematic review and metaanalysis of observational data, pooled incidence rates during pregnancy, postpartum period, and pregnancy and/or postpartum periods) were 5.1 (95% CL: 3.4 -6.7), 2.7 (95% CI: 1.6 -3.8), and 4.1 (95% CI: 3.1 -5.1), respectively 19 . Because early HIV infection is associated with increased HIV-1 viraemia 20, 21 , incident maternal infections might increase mother-to-child HIV transmission. Peri-conception periods, when condom use is reduced, are periods of high HIV-1 risk and women continue to be at high HIV-1 risk if condom use is not resumed during pregnancy and lactating periods. Antiretroviral-based interventions including PrEP have the potential to impact the HIV-1 risk in these periods of heightened risk in women and their use is likely to continue through postpartum breast feeding.

Tenofovir use during pregnancy and infant outcomes
Clinical trials of PrEP excluded pregnant women and women who became pregnant during the study stopped study drug. In an early drug development study involving infant macaque model of pediatric human immunodeficiency virus (HIV) infection, Van Rompay et al using doses of reverse transcriptase inhibitor 9-[2-(phosphonomethoxy) propyl] adenine (PMPA; tenofovir) ranging from 4 to 30 mg/kg of body weight administered subcutaneously once daily to 39 infant macaques for a short period of time (range, 1 day to 12 weeks), no adverse effects on infant macaques health or growth were observed. 22 Moreover, even chronic administration of a low dose of PMPA (10 mg/kg) starting at birth was not associated with any adverse health effects after 5 years of daily treatment 22 . Data on PrEP use during pregnancy available from HIV-1 uninfected women who used PrEP during peri-conception periods and early in the first trimester of the Partners PrEP Study shows no statistically significant differences in pregnancy and infant outcomes for those exposed to PrEP versus placebo (Table 4), including infant length, weight, and head circumference over one year of follow-up after birth. 23 In addition, there is safety data on HIV-1 infected women who have used FTC/TDF during pregnancy. In the large DART trial of routine vs. clinically-driven laboratory monitoring for HIV-1 care, Gibb et al. compared birth and growth outcomes between infants with 0% of in utero time with tenofovir exposure versus ≥90% of in utero time with tenofovir exposure. 24 After following children for at least 12 months (median follow up time=25 months), there were no differences in renal function, weight or height among tenofovir exposed versus unexposed children. Another source of data from a large US Antiretroviral Pregnancy Registry on 1,219 pregnancies with first-trimester exposure to the FTC/TDF have shown no increase in overall birth defects compared with the general population rates 25 .
Taken in aggregate, these data suggest that short-term PrEP is safe in pregnancy and FTC is similar to 3TC which has safety record for during pregnancy and postpartum breast feeding but the data on tenofovir are largely limited to teratogenicity and most did not directly assess PK and safety (given safety profiles among adults), among infants exposed to tenofovir in utero. Specifically, the data are incomplete for infants of HIV-1 uninfected lactating women using PrEP where tolerance/toxicity and not teratogenicity is the main focus.

Rationale for evaluation of PrEP among breastfeeding women
Pregnancy and postpartum breast feeding represent periods of heightened HIV-1 risk for women and for their infant if acute HIV-1 infection occurs 8,[15][16][17][18]26 . Women at the greatest risk for HIV-1 acquisition are in their child-bearing years, during which they face HIV-1 acquisition risk in settings where fertility is high such as Sub-Saharan Africa. After demonstration of PrEP efficacy and safety in the general population, study of the safety of PrEP use during lactating period is needed to inform guidelines and recommendations for PrEP use by lactating women. The benefits of breastfeeding on infant morbidity and mortality are well documented 27, 28 ; closely associated with survival and early cessation of breastfeeding is associated with increased infant mortality and morbidity in many developing countries. 27-31 Antiretroviral-based interventions including PrEP are powerful novel tools that could impact HIV-1 risk during pre-conception, pregnancy and lactating periods 1-4, 11, 12 . However, maternal adherence to PrEP medication may be compromised for fear of exposing their infant to the medication through the breast milk. In CAPRISA 004 11 , Mathews et al reported that women with pregnancies (n= 53) were less adherent to study product (1% Tenofovir Vaginal Gel or placebo) with median adherence of 50% compared to women without pregnancies (n =815) with median adherence of 60% (adjusted odds ratio= 0.52, 95%CI 0.41-0.66, p<0.0001) 32 . Current guidelines for PrEP use by pregnant and lactating women are compromised by the paucity of data, as clinical trials of PrEP excluded pregnant and breastfeeding women. Experience with tenofovir pharmacokinetics in pregnancy and postpartum breast feeding outside of clinical trials has largely been among HIV-1 infected women for PMTCT but because a majority of these infants were exposed to circulating drugs via the placenta and in addition to oral TDF, 33 their pharmacokinetic parameters likely represent a combination of both vertical-and oral-administration pharmacokinetic patterns. Like other nucleotide analog reverse transcriptase inhibitors (lamivudine and zidovudine), 34,35 both emtricitabine and tenofovir, have been shown in small studies to be excreted into breast milk.
In a previous study of the transfer of tenofovir in breast milk of two rhesus macaques, tenofovir was detected in the milk of both animals, but the peak concentrations (~0.6 to 0.8 μg/ml) were only ~2 to 4% of those detected in serum. 36 In the ANRS 12109 TEmAA (Tenofovir/Emtricitabine in Africa and Asia) study among five Ivorian mothers who chose to exclusively breast feed their infant and were administered one tablet of nevirapine (200 mg) plus two tablets of TDF (300mg)-FTC (200 mg) at the start of labor and one FTC/TDF daily tablet for 7 days postpartum, median tenofovir and emtricitabine breast milk doses represented 0.03% and 2%, respectively, of the proposed oral infant doses. 37 In that study, plasma neonatal tenofovir/emtricitabine levels were estimated by simulation from predicted tenofovir and emtricitabine breast milk doses and were not directly measured. However, it is difficult to predict drug transfer into milk based only on physicochemical properties and current models are limited in prediction of milk concentrations of drugs in humans. 38

Summary of rationale for the study
The standard for use of drugs during pregnancy and lactation is very high due to safety concerns for unborn or breasting infant. The primary goal of this study is to quantify the magnitude of FTC/TDF that breast feeding infants are exposed to when it is used as pre-exposure prophylaxis by their HIV-1 uninfected mothers. The primary measure will be infant plasma drug concentration. Women are at the greatest risk for HIV-1 acquisitions are in their child-bearing years, during which they face HIV-1 acquisition risk. A serious consequence of HIV-1 acquisition during pregnancy, delivery, and postpartum breastfeeding is vertical transmission of HIV-1 to their child because of high viraemia associated with acute HIV-1 infection. Therefore women are natural target population for pre-exposure prophylaxis to reduce HIV-1 risk during periods of heightened risk including lactation. Inadvertently, nursing infants for HIV-1 uninfected women taking PrEP for HIV-1 infection will be indirectly exposed to FTC/TDFvia breast milk. Transmission of biologically significant drug concentrations to nursing infants through breast milk could occur. A common reason for the cessation of breastfeeding is the use of medication by the nursing mother and advice by her physician to stop nursing. This study is intended to supply the pediatrician, obstetrician, and family physician with data concerning the excretion of FTC/TDF into human milk and absorbed by the nursing baby. This information is important not only to protect nursing infants from untoward effects of maternal FTC/TDF medication but also to allow effective pharmacologic chemoprophylaxis of breastfeeding mothers to reduce their HIV-1 risk. The benefits of breastfeeding are well known and undisputed, so clinician so clinicians should counsel mothers on both the clinical benefits and potential adverse effects to them and their infants before any recommendation for weaning. Thus, data gained from this study will permit a thorough evaluation of the benefits and risks of maternal FTC/TDF PrEP use during lactation.
Although current aggregated data on FTC/TDF safety show the frequency of adverse effects among infants exposed to FTC/TDF during pregnancy are similar to the general population rates, these data are incomplete for HIV-1 uninfected women using FTC/TDF PrEP to prevent acquisition of HIV-1 and are largely limited to fetal teratogenicity and not toxicity. Specifically, there are no published data from infants who have been breastfed while the HIV-1 uninfected mothers have taken FTC/TDF PrEP. The proposed study will provide the first empirical data on the magnitude and extent of infant absorption of FTC/TDF via breast milk when taken as PrEP by lactating women.

Overall Design
This is an interventional, open-label, single-arm, short-duration, repeat-dose pharmacokinetic study of daily FTC/TDF PrEP among HIV-1-uninfected lactating mother-infant pairs. Women and infants will be exposed to 10 days of FTC/TDF PrEPsufficient to reach steady-state but discontinuing thereafter. The overall goal is to quantify the magnitude and degree to which breastfeeding infants are exposed to FTC/TDF when used as PrEP by HIV-1-uninfected lactating women. We will conduct quantitative measurements and analyses of infant plasma drug concentrations, infant-plasma to breast-milk and breast-milk to maternal-plasma drug concentration ratios to characterize FTC and TDF transmission to breast feeding infants.

Aim To quantify the steady state concentrations of tenofovir (TFV) and emtricitabine (FTC) in infant plasma after FTC/TDF PrEP exposure through maternal breast milk.
Decisions about the safety of breast feeding during maternal ingestion of drugs require knowledge of the amount of drug which might be present in the milk.
Understanding the concentration of FTC/TDF infants are exposed to and subsequently absorbed via breast milk when taken as PrEP by lactating women is needed to permit a thorough evaluation of the benefits and risks of maternal FTC/TDF PrEP use during lactation. We will measure infant plasma, maternal plasma, and breast milk FTC and TFV concentrations in an open-label, repeat dose study of short-term daily FTC/TDF PrEP among HIV-1-uninfected lactating motherinfant pairs.
Hypothesis: Steady state TFV and FTC concentrations will be substantially low in infant plasma after PrEP exposure through maternal breast milk.
The primary outcomes of this aim will be the steady state infant plasma FTC and TFV levels, infant plasma to maternal breast-milk FTC and TFV concentration ratio, and maternal breast-milk to plasma FTC and TFV concentration ratio. A single infant plasma sample will be collected after the maternal 7 th PrEP dose. Maternal plasma and breast-milk samples for trough and peak drug levels will be sampled immediately before administration of the 7 th and 10 th doses and 1-2 hours after the 7 th and 10 th doses, respectively.

Population
HIV-1-uninfected mother-infant pairs will be recruited from the Partners PrEP Study sites in Kampala, Uganda and Thika, Kenya. The sample size of up to 50 pairs will be achieved across the two study sites, with numbers per site to be determined based on rate of recruitment and other study performance metrics. We anticipate that 25-30 mother-infant pairs (i.e., approximately half, for this two-site study) will be recruited at each site.

Eligibility
For infant's mother and father -Able and willing to provide informed consent for the infant to participate in the study -Of legal age ≥18 years to consent For HIV-1 uninfected mother, in addition to the criteria noted immediately above: -Willing to provide breast milk samples and breastfeed during the duration of the study 0-24 weeks postpartum -Breastfeeding an infant -HIV-1 uninfected based on negative HIV-1 rapid tests, both at study screening and at the enrollment visit -Adequate renal function, defined by normal creatinine levels and estimated creatinine clearance ≥60 mL/min -Not infected with hepatitis B virus, as determined by a negative hepatitis B surface antigen test -Not currently using PrEP -Enrollment of individuals with active and serious infections or active clinically significant medical problems will be at the discretion of the site investigator -Note: single mothers are eligible to participate in this study. Where possible the father's permission will be obtained. When the father is unknown, incompetent, deceased, or not reasonably available, or when only the mother has the legal responsibility for the care and custody of the child, the infant participant will be based on the mother's consent and documentation will be added to file For infant -Infant born to eligible women -Age 0-24 weeks -Otherwise infant has no serious infections or active clinically significant medical problems Exclusion criteria -Women breastfeeding more than one child -Preterm babies or infants with low birth weight (i.e. ≤2000mg)

Sample size
Up to 50 mother-infant pairs are expected to participate in this open-label, repeat-dose study. Sampling will be stratified on infant age: 0 to <12 weeks and 12 to 24 weeks, with up to 25 motherinfant pairs per age stratum. The proposed sample size is sufficient to permit a thorough evaluation of infant drug exposure while capturing changes in breast milk composition over varying postpartum weeks as well as changes in infant feeding, absorption, and clearance patterns -factors which contribute to the time-and phase-dependent variation of drug excretion into milk and subsequent infant absorption. The proposed sample size is consistent with samples sizes used in similar antiretroviral pharmacokinetic studies. 34

Ethical requirements for studies involving lactating women and infants
In the proposed study, the likelihood of serious risks are expected to be minimal for this 10 day study and the study is likely to yield generalizable knowledge beyond the study period. The Postpartum breastfeeding is a time of heightened HIV-1 acquisition risk and women infected during postpartum breastfeeding have increased risk of transmitting HIV-1 to their infant during acute infection 8,[15][16][17][18]26 . FTC/TDF was FDA-approved for use by HIV-1 infected individuals in 2004 following standard protocols for safety testing and in 2012 for use by HIV-1 uninfected individuals 5 . Increasingly, TDF and FTC are becoming an integral part of the antiretroviral regimens used in pregnant and post-partum women in many countries. Evidence from randomized clinical trials of HIV-1 uninfected women with first trimester exposure to TDF and FTC show no risk of birth defects or poor growth outcomes. 23 In addition, data from a large US Antiretroviral Pregnancy Registry on 1,219 pregnancies with first-trimester exposure to the FTC/TDF have shown no increase in overall birth defects compared with the general population rates 25 . Two studies have attempted to assess drug levels in infants specifically from breast milk exposure from their HIV-1 infected mothers. First, the ANRS 12109 TEmA study 37 enrolled five Ivorian HIV-1 infected women who chose to exclusively breast-feed their infants and were administered one tablet of Nevirapine (200 mg) plus two tablets of TDF (300 mg)-FTC (200 mg) at the start of labor and one FTC/TDF FTC/TDFdaily tablet for 7 days postpartum. Median TFV and FTC breast milk doses represented 0.03% and 2%, respectively, of the proposed oral infant doses. However, infant plasma drug levels were only estimated by simulation and not directly measured. Some of the model assumptions used in that study may be unrealistic and FTC/TDF was not at steady state. Second, the HPTN 057 protocol 39 , a multicenter study of the international maternal pediatric adolescent AIDS Clinical Trials Group (IMPAACT), administered 600 mg doses TDF in labor with no infant dosing to HIV-1 infected women. The goal was to assess fetal and newborn TDF exposure from labor through the end of the first week of life to protect against intrapartum and early postpartum/breast feeding HIV transmission. However, because the infants were exposed both transplacentally and through breast milk, the observed drug levels are likely a combination of pharmacokinetics of both routes. Importantly, the study only used TDF as single dose and not FTC/TDF. Our study is different from HPTN 057 protocol, in that we will use FTC/TDF and drug levels will be assessed after achieving steady state in the mothers at which drug absorption and elimination will be at equilibrium. Thus, this study will offer complimentary and new information. Additional studies have also assessed the FTC/TDF drug excretion in context of PTMC including Flynn et al 33 that evaluated the pharmacokinetics and safety of single-dose FTC/TDF in HIV-1-Infected pregnant women and their Infants. However, the focus in these trials were transplacentally transferred drugs and not through breast milk. Additional limitation for extrapolation of these data is that there is differential clearance of plasma TFV/FTC between that transferred across the placenta and doses received orally through breast milk because of the way gastric pH affect drug bioavailability. There has been extensive use and study of TDF (and FTC) in pregnant and breastfeeding HIV-1 infected women, including the recently-published results from the DART trial 40 . Thus, data from HIV-1 infected women provide important background for the proposed study, more than sufficient to permit the proposed work to be done safely. However, data from HIV-1 infected women, who may have different metabolism or excretion of medication, given disease and concurrent antiretroviral medications, mandate assessment of this important issue in HIV-1 uninfected women and their infants. The involvement of these vulnerable populations is critical to answer the research questions posed. Any benefits derived from this study are likely to have most impact on the population from which these women and infants are drawn. Research among pregnant or lactating women and children requires special ethical considerations.
For research to move forward in logical, stepwise increments, a framework has recently been described to identify specific steps needed to move clinical research into populations of pregnant and lactating women (Table 5). 41 For FTC/TDF PrEP, the next incremental step for safety assessment to inform the risks and benefits of PrEP use in lactating women is a time-limited evaluation of medication excretion in breast milk and infant absorption. Given the high risk of HIV-1 acquisition during pregnancy and postpartum breastfeeding and risk of transmission to the nursing infant in the event of acute HIV-1 seroconversion as well as the difficulties of condom use negotiation for women, the high efficacy of FTC/TDF in reducing HIV-1 acquisition combined with an excellent safety profile, Most NNRTIs are approved for use by HIV-1 infected women, including use during pregnancy and lactation; only TDF/FTC is approved for use by HIV-1 uninfected women to protect against HIV-1 infection 5. Are there known of potential risks to the mother or fetus?
Data from inadvertent exposure during first trimester do not demonstrate any risks 6. Are there known potential benefits to the mother or fetus?
Yes. The mother and infant will benefit from protection against HIV-1 infection 7. Can a case be made for the prospect of direct benefit to the mother or fetus?
Yes. Postpartum lactation is a time of heightened HIV-1 acquisition risk. Women infected during postpartum breastfeeding have increased risk of transmitting HIV-1 to their infant during acute infection. 8. Do alternatives exist to use of this drug in pregnancy?
No. This is the only drug currently approved for use among HIV-1 uninfected women for HIV-1 prevention 9. Which trimester(s) would be exposed with use Infants will be exposed during breast feeding. No trimester will be exposed as pregnant women will not participate. 10. How is the drug eliminated?
The drug is eliminated through the kidney and creatinine clearance is recommended to be calculated prior to initiating therapy. 11. Are clinical data available on pregnancy outcomes following exposure, and what is the quality of this evidence?
a) High quality evidence from randomized clinical trials of HIV-1 uninfected women with inadvertent first trimester exposure to TDF shows no risk of birth defects of poor growth outcomes. b) Evidence from observational cohort studies among HIV-1 infected women using tenofovir for HIV-1 treatment have shown no clinically significant growth delays in infants exposed to tenofovir in utero.
indicates that FTC/TDF PrEP is a promising intervention for HIV-1 prevention among lactating women, and warrants evaluation of breast milk FTC and TDF concentrations and absorption among infants.

Recruitment
Accrual will continue until up to 50 evaluable mother/infant are recruited into the study overall. Recruitment will be over in 6 months. A fully evaluable mother-infant pair is defined as one for which a complete set of specimen is obtained as specified in procedures and table 6. Motherinfant pairs that are not fully evaluable are technically not 'replaced' in the study, as all participants exposed to the study drug will remain in the study for safety monitoring and complete follow-up as originally scheduled, even if dosing is discontinued early; however these mother infant pairs will not count toward the targets sample size. The Thika, Kenya and Kampala, Uganda study sites have established local recruitment and screening methods that ensure efficiency in identifying research participants for the local study setting and target study population, including for research involving infants. The Thika, Kenya and Kampala, Uganda sites staff are highly trained to recruit from communities their research clinic are based in and are experienced in both individual and couples counseling. Recruitment strategies will include partnering with antenatal, postnatal, and immunization clinics, and other community-based organizations for referral of potential participants. Mother-infant pairs may also be recruited for possible inclusion in the study from other activities conducted at the Thika, Kenya and Kampala, Uganda study sites. Trained study staff will approach potential participants that may have been identified at the collaborating organizations.
All efforts will be made to maintain potential participant's privacy and confidentiality and to minimize potential coercion or the appearance of coercion. Contact persons at the collaborating organizations will be trained on aims of study and in handling of potential study participants. All screening procedures will be conducted at the study clinic. Trained study staff will inform the potential participants that their participation is completely voluntary, and that they will continue accessing care at their regular health facility if they choose not to participate in this study. Approval for all the recruitment procedures and materials will be sought from all relevant IRBS.
The screening process will proceed in a step-wise manner until either all screening procedures are completed or the mother-infant pair is determined to be ineligible. There is no time limit on the screening process.
For the mother-infant pairs found to be eligible for the study, informed consent for study participation and enrollment in the study may proceed on the same day when eligibility is determined.

Study procedures
Specific study procedures are detailed in Table 6. Visits will take place at screening and enrollment, and then daily thereafter, for up to 10 days.

Procedure for HIV-1 uninfected women
At screening, demographic, and breastfeeding information will be collected along with laboratory results to establish participant eligibility (serum creatinine, hepatitis B surface antigen, and HIV-1 rapid testing according to national algorithms).
At enrollment, HIV-1 testing will be performed for women to confirm eligibility (HIV-1 seronegative at the time of study start). HIV-1 uninfected women who have symptoms potentially consistent with acute HIV-1 infection (fever, rash, pharyngitis) will have enrollment deferred for 2 weeks at which time repeat serologic testing will be performed (and, if positive, will result in study exclusion). HIV-1 uninfected women will have a pregnancy test to confirm eligibility (must be not pregnant to complete enrollment).
At enrollment and then each daily follow-up visit, study mothers will be prescribed oral directly observed therapy (DOT) FTC/TDF PrEP. Participants will be counseled (for compliance /adherence to study procedure, drug toxicity, risk reduction) and complete breast feeding and medical history interviews with staff counselors.
During daily follow-up, HIV-1 uninfected women will complete a short quantitative interview to capture breasting pattern of the infants and proportion of infant feeding due to breast milk, adverse events, and concomitant medication use. Maternal blood and breast milk samples for drug assays will be collected at the 7 th and 10 th FTC/TDF dose (details in sample collection section below).
All data collections will be conducted in a private room in the participant's preferred language, according to the participant's fluency and preference. Development of data collection tools has been guided by our experience collecting clinical data for the Partners PrEP Study at multiple sites in Kenya and Uganda.

Participant retention and withdrawal
For this pharmacokinetic study, retention efforts will strive to achieve maximum compliance to study visit schedules to ensure the highest possible adherence to study medication. The Thika, Kenya and Kampala, Uganda sites are highly experienced in research participant follow-up and have developed retention methods tailored to and most efficient for their local study settings. Retention activities will include explanation of the study visit schedules and procedural requirements during the informed consent process and re-emphasis at each study visit, collection and updating of locator information, and use of appropriate and timely visit reminder mechanisms (including phone calls and text messages), and home visits for participant. To provide complete information at the end of the study, efforts will be made to have a final follow-up visit for each participant.
Participants may voluntarily withdraw from the study for any reason at any time. The site Investigator also may withdraw participants from the study in order to protect their safety and/or if they are unwilling or unable to comply with required study procedures.

HIV-1 testing
HIV-1 antibody testingfor women at screening and enrollment, will be performed in line with national HIV-1 testing algorithms for Kenya and Uganda. At enrollment, prior to maternal FTC/TDF dosing, the Infant will have an antibody test using parallel rapid HIV-1 tests in accordance with the national algorithms Kenya and Uganda. For mothers found to be infected with HIV-1 at screening, their infants will have a PCR to test for HIV-1 infection.

Sample collection
The frequency and volume of blood draws are defined in Table 6. Blood and breast milk specimens will be collected as per the study procedures manual.

ADMINISTRATIVE AND REGULATORY PROCEDURES
Obtain informed consent X X Apply inclusion/exclusion criteria X X Collect/update locator information X X X X X X X X X X X Collect demographic information X

COUNSELING
Provide HIV-1 pre and post-test counseling X X Adherence/compliance counseling X X X X X X X X X X Drug toxicity assessment and concomitant medication counseling X X X X X X X X X X

BREAST FEEDING DATA COLLECTION
Collect breastfeeding information X X X X X X X X X X X

LOCAL LABORATORY PROCEDURES
Creatinine and creatinine clearance X X HIV-1 serology (rapid tests) Infant HIV antibody test at enrollment only. X X HBV surface antigen X Urine pregnancy test X respectively. In total, four blood samples and breast milk samples each will be collected, two of respective sample at study visit 7 and study visit 10.

Procedures for the infant
The infant will be required to attend at least two study visits including the enrollment visit and the mother's study visit 7. However, mothers will be encouraged to come with their babies at their scheduled visit or if they feel the infant is unwell.
At the enrollment visit, we will obtain a small blood sample by a needle prick on the infant's finger or heel for an HIV-1 antibody test. The study staff will talk with the mother about the HIV-1 test and what it may mean to know the infant's HIV-1 status. Although it is very unlikely for the infant to be HIV-1 infected if their mother is HIV-1 uninfected, on very rare occasions it may be possible for the infant to have HIV-1 exposure from procedures involving medical injections or blood transfusion, or breastfeeding from a woman other than the mother. For infants found to be antibody positive for HIV-1, study staff will counsel the mother. The infant will be referred to the appropriate health facility for further check-up and no further study procedures will be performed on the mother and the infant.
At the mother's study visit 7, one blood sample [maximum 2 mL] will be collected from the infant to measure the infant plasma drug concentrations ingested via maternal breast milk. The blood sample shall be collected within 2 hours of study mother's 7th study drug dose (after maternal drug steady state). At each of the infant's scheduled study visits study staff will perform a clinical assessment. Infant age, gender, and weight will be recoded. If for any reason, infant blood sample cannot be collected on study visit 7, the sample can collected at any visit between visit 7 and visit 10 inclusive but before the end of the study. Table 6 shows details of all planned laboratory evaluations, including the relevant objectives, sample, responsible laboratory and the time of sample collection mother-infant pairs.

Laboratory Procedures
Procedures for specimen collection, transport, processing, storage and shipping will be included in the study procedures manual. All specimens should be transported to the laboratory within 4 hours of collection. All specimens will be labeled with a unique laboratory accession number which can be linked to the study unique coded identification number. Plasma and breast milk samples will be processed and stored at -70°C and subsequently analyzed by Johns Hopkins Clinical Pharmacology Laboratory, USA. Tenofovir and emtricitabine concentrations will be measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) previously validated for human plasma and breast-milk. [42][43][44] The analytic laboratory conforms to international standards of good laboratory practices (GLP) and GLP-like analyses for clinical samples.

Training and Quality Assurance
Demographic, breast feeding, and clinical data will be entered onto standard case report forms. Study staff involved in obtaining informed consent and client interviews, clinical examinations and specimen collection will receive relevant training prior to the commencement of this study. Pre and post training assessments will be conducted. Senior study staff members will continue to review clinical technique and assessment over the study period.

Participant retention and withdrawal
For this pharmacokinetic study, the goal is to achieve high FTC/TDF adherence to reflect maximum possible exposure from standard FTC/TDF PrEP doses. The project will recruit until up to 50 evaluable mother-infant pairs successfully complete all study procedures.
Participants may voluntarily withdraw from the study for any reason at any time. The site Investigator also may withdraw participants from the study in order to protect their safety and/or if they are unwilling or unable to comply with required study procedures. Reasons for withdrawal will be recorded.

PrEP medication
This study will provide open-label PrEP to HIV-1 uninfected lactating women. Only eligible HIV-1 uninfected women will receive study drug in this study. Tenofovir disoproxil fumarate and emtricitabine are reverse transcriptase inhibitors that have been approved for the treatment of HIV-1 infection in humans in Kenya, Uganda, and the United States. A fixed-dose, oral coformulation of FTC/TDF will be used in this study. The dose of FTC/TDF is the standard dose approved by the U.S. Food and Drug Administration. PrEP will be prescribed for once-daily oral 200m FTC/300mg TDF by directly observed therapy (DOT). Counseling on the medications being used, their side effect profiles, contraindicated concomitant medication, what to do if side effects are experienced will be done. The pharmacy and storage facility will have locked, climatecontrolled environments, with controlled temperature to remain within limits allowed by the manufacturer for drug storage. Study medication will be donated by Gilead Sciences.

Adherence
High adherence is critical for PrEP effectiveness in preventing HIV-1 acquisition and understanding the magnitude infant drug exposure for tolerance/toxicity assessment. Daily oral 200mg FTC/300mg TDF PrEP will be administered by directly observed therapy (DOT) to study women at the study site. Brief adherence and compliance counseling will be provided at each scheduled visit.

Discontinuation of PrEP
Use of PrEP may be interrupted by the site Investigator due to safety concerns for the participants (mother and infant), use of concomitant medications that could interfere with PrEP or present a safety concern, or if the participant is unable or unwilling to comply with study procedures. All treatment interruptions will be documented.
Concomitant medication use will be recorded. PrEP will be avoided, per investigator discretion, in individuals receiving ongoing therapy, interferon (alpha, beta, or gamma) or interleukin (e.g., IL-2) therapy, metformin, systemic aminoglycoside antibiotics, amphotericin B, cidofovir, systemic chemotherapeutic agents, other agents with significant nephrotoxic potential, other agents that may inhibit or compete for elimination via active renal tubular secretion (e.g., probenecid).

IV. SAFETY
The Partners PrEP Study demonstrated that PrEP (including FTC/TDF) was safe for use in heterosexual men and women from Kenya and Uganda. There were no statistically significant differences in the frequency of deaths, serious adverse events, adverse events overall or key laboratory adverse events (specifically, creatinine elevation and phosphorus decrease) for those receiving PrEP compared to those receiving placebo.
All adverse events, for both mother and infant participants will be documented. The severity of clinical symptoms will be scored using the DAIDS Table for Grading the Severity of Adult and Pediatric AEs 45 . Decisions to hold PrEP due to clinical and/or other laboratory safety reasons will be at the discretion of site Investigator. Adverse events will be reported according to relevant IRBs policies and regulations.
Clinical symptoms will be systematically assessed in a structured medical history administered to mother participants. Clinical side effects of TDF and FTC/TDF that have been reported are primarily gastrointestinal, including nausea, vomiting, and flatulence.

V. DATA ANALYSIS
The goal of the analysis is to quantify the magnitude and level of infant exposure to FTC/TDF PrEP through maternal breast milk. Data will be summarized in tabular and graphical forms for presentation. Descriptive statistics will be calculated for demographic and clinical data for mothers and infants. Categorical variables will be summarized as frequencies with proportion, and for continuous measures using mean and standard deviations or medians and ranges, as appropriate. The magnitude of infant exposure to FTC/TDF PrEP will be quantified using infant plasma drug concentrations, infant plasma-to-breast milk and breast milk-to-maternal plasma drug concentration ratios. Antiretroviral concentrations and milk-to-plasma ratios data will be presented as medians and ranges. Additionally, drug exposure index will be computed to estimate the infant daily drug exposure from breast milk feeding.

VI. HUMAN SUBJECTS CONSIDERATIONS
The study protocol, site-specific informed consent forms, participant comprehension questionnaire, education, and recruitment materials, and other requested documentsand any subsequent modificationswill be reviewed and approved by the IRBs/ECs responsible for oversight of research conducted at the study sites. Subsequent to initial review and approval, the responsible IRBs/ECs will review the study at least annually.

Informed consent
Written informed consent will be obtained from each study mother prior to both screening and enrollment. Potential research subject's comprehension of study procedures will be assessed by trained study staff performing the consent process using a comprehension questionnaire. Potential participants don't need to answer all questions correctly to enroll. If in the opinion of the study staff the participant demonstrates poor or inadequate understanding of the study procedures after further explanation and repeat assessment, study procedures may be deferred to another visit and subsequent participation in the study will be at the discretion of the site principal investigator. The fathers' permission will be obtained, where possible. When the father is unknown, incompetent, deceased, or not reasonably available, or when only the mother has the legal responsibility for the care and custody of the child, the mother's consent will be used and documentation will be added to file. Participants will be offered copies of the informed consent forms.
Each study site is responsible for developing study informed consent forms for local use that describe the purpose of screening and of the study, the procedures to be followed, and the risks and benefits of participation, in accordance with all applicable regulations, based on the schedule of procedures in Table 6. Each site also is responsible for translating the forms into local languages and verifying the accuracy of the translation by performing an independent backtranslation. After the local IRBs approvals, consents forms will be returned to UW IRB for final approval and stamping. Study procedures will not begin before the final approvals are obtained from all the relevant IRBs.

Risks
Participants may experience discomfort or pain when undergoing phlebotomy. They also may feel dizzy or faint, and/or develop a bruise, swelling, or infection where the needle is inserted.
Participants may become worried or anxious while waiting for their HIV-1 test results. Participants who learn that they have HIV-1 may experience anxiety or depression related to their test results. At the study sites, HIV-1 counseling will be provided by counselors and clinicians who have been trained in specific issues, including stigma, blame, methods to avoid transmission, and available support services.
Although study sites will make every effort to protect participant privacy and confidentiality, it is possible that participants' involvement in the study could become known to others, and that social harms may result (i.e., because participants could become known as participating in a trial involving medication used for HIV-1 treatment). For example, participants could be treated unfairly or discriminated against, or could have problems being accepted by their families and/or communities.
Risks and side effects related to PrEP include: occurring in a minority of individuals taking PrEP -gastrointestinal intolerance, such as nausea, diarrhea or vomiting, flatulence; rare but seriouslactic acidosis/ severe hepatomegaly with steatosis, renal impairment, including cases of acute renal failure and Fanconi's syndrome (renal tubular injury with severe hypophosphatemia), increase in bone metabolism leading to osteopenia, hypersensitivity reaction. HIV-1 resistance may also emerge in some individuals with undiagnosed primary HIV-1 infection taking only FTC/TDF PrEP because FTC/TDF alone does not constitute a complete regimen for HIV-1 treatment. HIV-1 uninfected subjects who have symptoms potentially consistent with acute HIV-1 infection (fever, skin rash, pharyngitis, fatigue, myalgia, etc) will have enrollment deferred for 2 weeks at which time repeat serologic testing will be performed and, if positive, will result in exclusion from study participation. Serious adverse reactions would be expected to be extremely rare with only 10 days of PrEP use. Risks to infants associated with exposure to FTC/TDF through breastfeeding are unknown but would likely be similar to potential risks in adults.

Benefits
There may be no other direct benefits to participants in this study. However, participants and others also may benefit in the future from information learned from this study. Because of the short duration of this study, a 10-day period of daily PrEP is not expected to offer adequate protection against HIV-1 acquisition to women. On average, at least 2 weeks of daily PrEP would be required to significantly reduce the risk of HIV-1 infection. Women will be counseled to continue using other HIV-1 prevention strategies including condoms and screening and treatment for sexually transmitted infections. All participants will be provided with HIV-1 prevention services.

Care for persons identified as HIV-1 infected at screening
This study may identify persons who are infected with HIV-1 as part of the study screening process. Study staff will provide participants with their HIV-1 test results in the context of post-test counseling. Persons identified as HIV-1 infected during the study screening process, will be referred to local HIV-1 care services and/or other agencies that provide care or access to treatment.

Treatment for injury
Participants will be asked to inform the study staff if they feel they or their infants have been injured because of taking part in the study. Injuries may also be identified during laboratory testing, medical histories, and physical examinations. Treatment for adverse events related to study participation will be provided by the study clinic. If treatment is required that is beyond the capacity of the study clinic, the study doctors will refer the participant to appropriate services or organizations that can provide care for the injury.

Study records
Site Investigators will maintain, and store in a secure manner, complete, accurate and current study records throughout the study. The investigator will retain all study records after completion of the study according to IRB/EC and national regulations. Study records include administrative documentation and regulatory documentation as well as documentation related to each participant screened and/or enrolled in the study, including informed consent forms, locator forms, case report forms, notations of all contacts with the participant, and all other source documents.

Confidentiality
Every effort will be made to protect participant privacy and confidentiality to the extent possible.
The study sites will establish a standard operating procedure for confidentiality protection that reflects the local study implementation plan and the input of study staff and community representatives to identify potential confidentiality issues and strategies to address them. In addition to local considerations, the protections described below will be implemented at all sites.
All study-related information will be stored securely at the local study sites. All participant information will be stored in areas with limited access. Data collection, administrative forms, laboratory specimens, and other reports will be identified only by a coded number to maintain participant confidentiality. All records that contain names or other personal identifiers, such as locator forms and informed consent forms, will be stored separately from study records identified by code number. All local databases will be secured with password-protected access systems. Forms, lists, logbooks, appointment books, and any other listings that link participant ID numbers to other identifying information will be stored in a separate, locked file in an area with limited access. The link between the participant's personal identifiers and the code on the participant's data and samples will only be kept at the study sites and only until December 31 st , 2015 when the study data collection is expected to be completed. After that time, this link will be destroyed.
Participants' study information will not be released without their written permission, except as necessary for oversight by:

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The study investigators or designees -Study funders -Applicable local authorities, e.g., Ministry of Health -Site IRBs/ECs -University of Washington -Any additional study sponsors

Study limitations
While infant feeding practices throughout Uganda and Kenya can be broadly characterized as being universal and predominantly breastfeeding of prolonged duration, factors potentially modified by infant feeding practices of rural versus urban population will need careful interpretation given the small sample size involved. The study population may be also expected to differ on some characteristic from the overall source population due to the exclusion of mothers with multiple live births and the need to live near the study site in order to be able to attend for frequent study visits. In addition, single sample designs can yield imprecise estimates because of random variation; however, this is especially important when the main goal of the study is hypothesis testing for detecting differences between groups. Despite these limitations described, the pharmacokinetic factors to be studied are likely to be robust to cultural and socio-economic differences amongst groups in population studied and those not studied.

Application of study results
This study is intended to provide the pediatrician, obstetrician, family physician and other primary healthcare with data concerning the excretion of FTC/TDF FTC/TDF into human milk and absorbed by the nursing baby. This information is important not only to protect nursing infants from untoward effects of maternal FTC/TDF FTC/TDF medication but also to allow effective pharmacologic chemoprophylaxis of breastfeeding mothers to reduce their HIV-1 risk. Data gained from this study will permit a thorough evaluation of the benefits and risks of maternal FTC/TDF PrEP use during lactation.

Dissemination of study results
Results from this study are not expected to have any direct implications for the subject's health care or for the health care of her baby. Final results that constitute clinically significant or scientifically relevant information will be made available to relevant target audiences. Primary target audiences include study subjects, women of reproductive age in Uganda and Kenya and their health providers. Information will be made available through appropriate local channels such as at academic and public health forums, medical and nursing professional societies, women's community groups and HIV related non-government organizations, ministries of health. Manuscripts will also be submitted for peer review.

Time line
The

Intellectual property rights
The study team jointly will own the intellectual property rights for this study.