Lopinavir/Ritonavir versus Lamivudine peri-exposure prophylaxis to prevent HIV-1 transmission by breastfeeding: the PROMISE-PEP trial Protocol ANRS 12174

Background: Postnatal transmission of HIV-1 through breast milk remains an unsolved challenge in many resource-poor settings where replacement feeding is not a safe alternative. WHO now recommends breastfeeding of infants born to HIV-infected mothers until 12 months of age, with either maternal highly active antiretroviral therapy (HAART) or peri-exposure prophylaxis (PEP) in infants using nevirapine. As PEP, lamivudine showed a similar efficacy and safety as nevirapine, but with an expected lower rate of resistant HIV strains emerging in infants who fail PEP, and lower restrictions for future HIV treatment. Lopinavir/ritonavir (LPV/r) is an attractive PEP candidate with presumably higher efficacy against HIV than nevirapine or lamivudine, and a higher genetic barrier to resistance selection. It showed an acceptable safety profile for the treatment of very young HIV-infected infants. The ANRS 12174 study aims to compare the risk of HIV-1 transmission during and safety of prolonged infant PEP with LPV/r (40/10 mg twice daily if 2-4 kg and 80/20 mg twice daily if >4 kg) versus Lamivudine (7,5 mg twice daily if 2-4 kg, 25 mg twice daily if 4-8 kg and 50 mg twice daily if >8 kg) from day 7 until one week after cessation of BF (maximum 50 weeks of prophylaxis) to prevent postnatal HIV-1 acquisition between 7 days and 50 weeks of age.Methods: The ANRS 12174 study is a multinational, randomised controlled clinical trial conducted on 1,500 mother-infant pairs in Burkina Faso, South Africa, Uganda and Zambia. We will recommend exclusive breastfeeding (EBF) until 26th week of life and cessation of breastfeeding at a maximum of 49 weeks in both trial arms.HIV-uninfected infants at day 7 (± 2 days) born to HIV-1 infected mothers not eligible for HAART who choose to breastfeed their infants.The primary endpoint is the acquisition of HIV-1 (as assessed by HIV-1 DNA PCR) between day 7 and 50 weeks of age. Secondary endpoints are safety (including resistance, adverse events and growth) until 50 weeks and HIV-1-free survival until 50 weeks.Discussion: This study will provide a new evidence-based intervention to support HIV-1-infected women not eligible for HAART to safely breastfeed their babies.Trial registration number (http://www.clinicaltrials.gov): NCT00640263. © 2012 Nagot et al.; licensee BioMed Central Ltd. We designed a multicentre randomised controlled pragmatic trial. Although double blinding is not implemented for this study, we will mask drug bottles with study labels and keep study physicians in charge of study visits and adverse events collection, and staff in charge of anthropometric measurements unaware of treatment allocation. In addition, biologists in charge of infant HIV diagnosis (primary outcome) will be blinded. We here describe the version 3.0 of the study protocol, registered at http://www.clinicaltrials.gov ({“type”:”clinical-trial”,”attrs”:{“text”:”NCT00640263″,”term_id”:”NCT00640263″}}NCT00640263). All women who agree to participate in the study will be counselled to exclusively breastfeed their babies up to 6 months of age, and to gradually introduce complementary food thereafter and to stop breastfeeding at 49 weeks. This will give women sufficient time to prepare for cessation of breastfeeding. Continuous counselling on a nutritionally balanced diet will be made available up to 50 weeks of age. In order to cover the whole breastfeeding period, the maximum duration of prophylaxis is 50 weeks. The infants will be tested for HIV-1 at Birth, Day 7, Week 6, 14, 26, 38 and 50 (Figure ​(Figure11). ANRS 12174 PROMISE-PEP trial design including scheduled follow-up visits. Infants will be recruited and followed in Ouagadougou (Burkina Faso), East London (South Africa), Mbale (Uganda) and Lusaka (Zambia). HIV-infected pregnant women will be identified at antenatal clinics. Breastfed infants of HAART-treated mothers ingest sufficient amounts of ART in milk to reach plasma therapeutic levels (at least for NVP, LPV/r, AZT and 3TC) [22]. Therefore, in order to avoid overdosage of LPV/r and Lamivudine in babies, our trial will only recruit babies born to mothers that are not eligible for HAART according to local recommendations, i.e. with >350 CD4 cells/μL blood. A baby will be included if she/he: • is a singleton • is breastfed at day 7 by her/his mother and her/his mother intends to continue breastfeeding for at least 6 months • has a post-partum blood sample with a negative HIV-1 DNA PCR test result at day 7 (+/− 2 days) • has received some antiretroviral prophylaxis at birth or during the first week • if the mother: has reached the local legal age for participating in medical research studies is HIV-1 infected (with or without HIV-2 infection) and is not eligible for HAART has received some antiretroviral prophylaxis during pregnancy or delivery, has an antenatal lymphocyte CD4 count above the threshold for HAART initiation in pregnant women according to the national recommendation in each site resides within the study area and is not intending to move out of the area in the next year gives consent to participate for her and her infant A baby will be excluded if: • S/he presents clinical symptoms and/or biological abnormalities equal to or greater than grade II of the ANRS classification for adverse event on the day of enrolment, with exceptions for haemoglobin (baby excluded if Hb < 12 g/dL) and absolute neutrophils count (baby excluded if neutrophils < 1200 cells/μL) • S/he presents with serious congenital malformation(s) • Her/his birth weight was < 2.0 kg • The mother has participated in the ANRS 12174 trial for a previous pregnancy • S/he and her/his mother are participating in another clinical trial on the day of enrolment • Acquisition of HIV-1 (as assessed by HIV-1 DNA PCR) between day 7 and 50 weeks of age. • Severe adverse events (SAE) grade III or IV possibly-related or with undetermined relation to the study drug (according to ANRS SAE reporting procedures), up to 50 weeks. • In vitro HIV-1 resistance to antiretrovirals in infants who will get infected with HIV-1 during the study • Growth, i.e. length and weight up to 50 weeks of age • HIV-1-free survival from day 7 until 50 weeks (event: infant death or acquisition of HIV-1 infection in infants) HIV acquisition is defined as follows: HIV-1 infection will be established if HIV-1 DNA is detected in blood (detection threshold of 150 viral copies/mL). All first positive HIV-1 DNA PCR will be confirmed by a second test on another sample taken 2 to 5 days later. If the second test is negative, then the child will be considered HIV-1 uninfected for this study visit. In order to ensure that infants infected with HIV-1 at week 6 (or at first follow-up visit if week 6 visit is missed) were not already infected at day 7, we will systematically and retrospectively re-test their dried blood spots (DBS) collected at day 7 using both a Roche-Amplicor HIV-1 DNA PCR and a real time HIV-1 RNA PCR (Figure ​(Figure1).1). In case of a positive result with at least one of the latter tests, the child will be considered HIV-1 infected at day 7 and therefore excluded from the trial. This situation is expected to occur for very few children. Infants fulfilling the inclusion criteria will be randomised on day 7 (+/− 2 days). Randomisation will be stratified by country using permuted blocks of randomly varying sizes 4 and 6. The eligible infants will be allocated to one of two arms: LPV/r (twice daily) or Lamivudine (twice daily) according to a 1:1 ratio. All randomisation lists will be prepared (and kept) by an independent statistician in Montpellier, as well as by the data monitoring committee (DMC) statistician. All antenatal clinics where the women will be screened benefit from a PMTCT programme including routine counselling and HIV testing. All HIV-infected pregnant women attending these ANCs with a gestation period between 28 and 40 weeks will be targeted for the first screening visit. After comprehensive counselling on infant feeding, mothers intending to breastfeed will be briefly informed about the study and invited to attend the first screening visit. After informed consent for this visit, women will be offered a CD4 cell count and clinical assessment (HIV-1 disease staging and investigation for opportunistic infections). All HIV-1-infected women and their babies will access PMTCT perinatal prophylaxis according to national guidelines [23]. Pregnant women will be encouraged to deliver at a maternity clinic. Pregnant women who require ART according to existing guidelines [23] will be referred to national ART access programmes in order to receive HAART for the remainder of the pregnancy, through delivery and continued life-long. She and her infant will not be eligible for the trial. The post-natal screening will be carried out from 1 to 6 days after delivery. The baby will be tested for HIV-1 infection using a real-time PCR DNA assay, and for biological abnormalities (full blood count, ALAT/ASAT, serum-creatinine). Finally, the study physician will assess the baby for clinical exclusion criteria. If these clinical criteria are met, the baby will be referred to the reference paediatrician for appropriate care. Systematic follow-up visits will be organised at week 2 and every 4 weeks until week 50 (final visit) (Figure ​(Figure2),2), with clinical examination, adverse event reporting, infant length/weight, counselling on infant feeding and drug adherence. The primary outcome (HIV-1 DNA) will be measured at week 6, 14, 26, 38 and 50. ANRS 12174 PROMISE-PEP trial screening, enrolment and follow-up visits contents. Mothers and babies will be followed by research/clinic-based visits, and if needed, by community-based visits. Children will be assigned to scheduled clinic visits and mothers will be encouraged to come back to the research site whenever they or their babies are sick. In babies, adverse events will be searched for and reported. Severe adverse events will be reported according to the sponsor guidelines. Infant feeding practices will be monitored using tools based on WHO guidelines [24]. Weight and length will be measured twice based on guidelines developed by the WHO and z-scores calculated using the new WHO child growth standards [25] and the free WHO program ( http://www.who.int/childgrowth/training/en/). The intervention will be stopped in children shown to be HIV-1-infected in order to reduce the period of unnecessary exposure to LPV/r or Lamivudine mono-prophylaxis and risk of emergence of resistant viral populations to a minimum. At 50 weeks of age, a final real time DNA PCR test will be done on all babies, in order to detect late HIV-1 transmission due to prolonged or resumed BF. Child survival will be assessed at 38 weeks and at 50 weeks of age. Hospital records and verbal autopsy will be used to measure overall and cause-specific infant mortality. Morbidity will be measured by recall within the last week of the visit, in-between visits and hospitalisations since last visit. The study treatments will be administered using commercial syrup formulations of lopinavir/ritonavir (80/20 mg/mL) or lamivudine (10 mg/mL). Study drug bottles of LPV/r and Lamivudine will be masked by study labels. Lopinavir/ritonavir (80/20 mg/mL) will be given to the baby twice daily as recommended in infants, with the following scheme: 40/10 mg (0.5 mL) twice daily if 2-4 kg and 80/20 mg (1 mL) twice daily if >4 kg. Similarly, lamivudine will be given as follows: 7,5 mg (0,75 mL) twice daily if 2-4 kg, 25 mg (2.5 mL) twice daily if 4-8 kg and 50 mg (5 mL) twice daily if >8 kg. These dosages were calculated on the basis of a pharmacokinetic study of LPV/r in young infants done in Paris [21]. For Lamivudine, calculations were based on a pharmacokinetic study carried out by Moodley et al. [26]. Infants whose mothers need and initiate HAART during the trial will be discontinued from the study medication in order to avoid excessive intake of ART in babies due to high diffusion of lamivudine in breast milk [27]. In addition, the maternal HAART regimen will much decrease the risk of HIV transmission through breast milk [9]. The primary outcome will be assessed using a superiority analysis. We expect the risk of acquiring HIV-1 infection between day 7 and 50 weeks of age to range between 3% and 5% in the lamivudine group, based on the findings of the MITRA study which reported a transmission of 1.2% (95%CI: 0 to 2.4) between 6 weeks and 6 months and association between CD4 count and risk of transmission observed in the VTS study [2]. This range of 3 to 5% accounts for population with CD4 count > 350 cells/μl and additional HIV transmission between day 7 and week 6, and between 6 months and 50 weeks. Considering its higher anti-HIV-1 activity, we expect a higher efficacy of LPV/r, ranging from 1 to 2.5%, which would be considered as a clinically relevant difference compared with the lamivudine arm. Table ​Table11 displays different scenarios, based on a comparison of proportions with 80% power, a two-sided alpha error of 5% and 10% lost to follow-up. Including 1,500 infants in the trial (750 in each arm) would allow us to cover most scenarios under the above hypotheses. Analysis methods will follow the CONSORT guidelines [28] (Figure ​(Figure3)3) and recommendations of the GHENT group related to the mother-to-child transmission studies [29,30] and breastfeeding patterns [31]. ANRS 12174 PROMISE-PEP trial, planned CONSORT patient flow chart. All tests will be two-sided. Descriptive results, efficacy and safety estimates and their corresponding 95% CIs will be presented. The statistical significance is set at p < 0.05. Provided there are no major differences of relative transmission risk between sites, the stratified (site-specific) randomisation will be adjusted for in the analyses. Moreover, potential confounders, such as maternal CD4 counts, will be considered for further adjustment if they are imbalanced at baseline, according to thresholds pre-defined in the plan of analysis. One interim analysis will be carried out by the DMC when about half of the projected HIV-transmissions have occurred. We will suggest that the DMC follows the DAMOCLES group recommendations [27]. Analyses for the primary endpoint will be undertaken on an intention-to-treat basis. – Uninfected drop-outs and deaths will be censored at the last outcome measurement. – Children of mothers initiating HAART during the study will no longer receive the study intervention. They will be followed up for all outcomes as the other children and will not be censored in the primary analysis. – Children who stop breastfeeding for various reasons (mother illness or death, etc.) will be withdrawn from study drugs one week later. They will continue follow-up until 50 weeks of age and will be included in the primary analysis. The main endpoint is acquisition of HIV-1 (as assessed by HIV-1 DNA PCR) between day 7 and 50 weeks of age, as defined previously. The primary endpoint will be analysed using a time to event approach. HIV-1 infections occurring between day 7 and week 50 will be compared between the two groups of treatment using proportional hazard modelling with hazard ratio and its 95% CI adjusted for site and any confounders. Relative treatment efficacy will be calculated as 100 × (1-HR) where HR is the hazard ratio of HIV-1 transmission in the LPV/r group compared to the Lamivudine group. The effect size will also be expressed as the absolute difference between risks with its 95% confidence interval. The reduction in the number of patients who need to be treated when given LPV/r compared to when given Lamivudine to prevent HIV-1 infection in one infant will be computed [32]. If LPV/r is not demonstrated to be superior to Lamivudine in the primary analysis, we will conduct a non-inferiority analysis of the primary outcome. This analysis is supported by the expectancy of a better resistance profile of LPV/r compared with Lamivudine. A 95% confidence interval (adjusted for interim analyses) for the hazard ratio of the primary efficacy outcome will be constructed. The upper boundary for the non inferiority is set at < 1.2, as we believe that any further reduced efficacy would have important public health consequences. We will also carry out per protocol analyses for this non-inferiority analysis. The conclusion will be drawn from both the intention to treat and the per protocol analyses. Under the closed-testing procedure with sequential testing of the superiority and non-inferiority analyses, the overall 2-sided type 1 error is maintained at 5%. All biological specimens will be analysed and stored at each study site. Paediatric HIV-1 infection will be diagnosed at each site on Dried Blood Spots (DBS) by means of real-time PCR, using a commercial kit (Generic HIV Charge Virale, Biocentric, France) [33]. When necessary (see Figure ​Figure1),1), a commercial real time RNA PCR (Generic HIV Charge Virale, Biocentric, France) as well as another commercial DNA PCR test (Roche Amplicor) will be used in the coordinating laboratory (Montpellier). The real-time PCR technique is implemented with the support of well-validated, standardised protocols designed by a working group of the French National Agency for Research on AIDS and Viral Hepatitis (ANRS) [34]. It is well adapted to the use of DBS collected on filter paper, an ideal tool for early paediatric HIV-1 diagnosis [35]. Maternal HIV-1 antibody detection will be performed by ELISA and/or rapid tests, according to each site’s national optimised algorithms [36]. Maternal plasma HIV-1 RNA quantification (“viral load”) will be measured by means of a commercially available real time RNA PCR test (Generic HIV Charge Virale, Biocentric, France) [33]. CD4 counts will be performed by flow cytometry. Haematological and biochemical assays will be performed on babies' plasma samples by means of already available automated procedures at the site laboratories. These tests will be carried out for safety monitoring, and will include full blood counts and blood concentration of liver enzymes. In addition, plasma LPV/r and Lamivudine will be detected in blood at weeks 6 and 38 by high pressure liquid chromatography (HPLC) (JM Tréluyer, Saint-Vincent de Paul Hospital, Paris). HIV resistance mutations to antiretrovirals will be identified by sequencing of the pol genes. Laboratory and quality control procedures will follow WHO Good Laboratory Practice Guidelines [37]. HIV-1 serology, CD4 counts, and haematology/biochemistry assays will be submitted to a stringent quality assessment programme, to an international external quality control programme and to intensive GLP training addressed to the laboratory personnel in all 4 sites. Real time PCR technology transfer and training will be implemented in all 4 sites with implementation of a quality assessment and quality control programme from the Montpellier team and the ANRS AC11 medical virology group. Ongoing training and monitoring will occur during the full duration of the follow-up period of the study. Likewise, initial and ongoing training and standardization will be undertaken to ensure adequate reproducibility and validity of anthropometric measurements. The study will be conducted in accordance with the sponsor’s (ANRS) Charter of Ethics, with the International Conference on Harmonisation (ICH) guidelines for good clinical practice and with the Medicines Control Council guidelines for good clinical practice in the conduct of clinical trials in human participants in South Africa. The study protocol has been submitted to and approved by the Ethical Committee for Health Research in Burkina Faso, the Biomedical research Ethics Committee in Zambia, the Uganda National Council for Science and Technology, the Stellenbosch University ethical committees, the Medicines Control Council in South Africa and the Regional Committee for Medical research Ethics of Norway. Written informed consent and assent will be obtained in vernacular language from the mother of participating mother-infant pairs. The informed consent process will be implemented with an independent third person (the ‘witness’) for illiterate mothers or when translation is required. This witness will sign the consent form together with the mother and the investigator. Mothers requiring HAART during pregnancy and infants diagnosed with HIV at screening or during the trial will be referred to the study-related HIV care unit for prompt HIV management according to local guidelines. In particular, infants will be treated with HAART as soon as HIV infection is diagnosed.