Efficacy and safety of azithromycin-chloroquine versus sulfadoxine-pyrimethamine for intermittent preventive treatment of plasmodium falciparum malaria infection in pregnant women in Africa: An open-label, randomized trial

Background The World Health Organization recommends intermittent preventive treatment in pregnancy (IPTp) with sulfadoxine-pyrimethamine (SP) in African regions with moderate to high malaria transmission. However, growing resistance to SP threatens the effectiveness of IPTp-SP, and alternative drugs are needed. This study tested the efficacy, tolerability, and safety of a fixed-dose combination azithromycin-chloroquine (AZCQ; 250 mg AZ/155 mg CQ base) for IPTp relative to IPTp-SP. Methods and Findings A randomized, Phase 3, open-label, multi-center study was conducted in sub-Saharan Africa (Benin, Kenya, Malawi, Tanzania, and Uganda) between October 2010 and November 2013. Pregnant women received 3 IPTp courses with AZCQ (each course: 1,000/620 mg AZCQ QD for 3 days) or SP (each course 1,500/75 mg SP QD for 1 day) at 4- to 8-week intervals during the second and third trimester. Long-lasting insecticide-treated bednets were also provided at enrollment. Study participants were followed up until day 28 post delivery (time window: day 28-42). The primary endpoint was the proportion of participants with sub-optimal pregnancy outcomes (a composite endpoint comprising live-borne neonates with low birth weight [LBW, <2,500 g], premature birth [<37 weeks], still birth [>28 weeks], abortion [≤28 weeks], lost to follow-up prior to observation of pregnancy outcome, or missing birth weight). The study was terminated early after recruitment of 2,891 of the planned 5,044 participants, due to futility observed in a pre-specified 35% interim analysis. In the final intent-to-treat dataset, 378/1,445 (26.2%) participants in the AZCQ and 342/ 1,445 (23.7%) in the SP group had sub-optimal pregnancy outcomes, with an estimated risk ratio (RR) of 1.11 (95% CI: 0.97, 1.25; p = 0.12). There was no significant difference in the incidence of LBW between treatment groups (57/1138 [5.0%] in the AZCQ group, 68/1188 [5.7%] in the SP group, RR 0.87 [95% CI: 0.62, 1.23]; p = 0.44). IPTp-AZCQ was less welltolerated in mothers than IPTp-SP. Occurrences of congenital anomalies, deaths, and serious adverse events were comparable in neonates for both groups. Limitations included the open-label design and early study termination. Conclusions IPTp-AZCQ was not superior to IPTp-SP in this study and alternatives for IPTp-SP remain to be identified. The proportions of sub-optimal pregnancy outcomes and LBW were lower than expected, which may be linked to insecticide-treated bednet use throughout the study. Reduced incidences of symptomatic malaria infection and peripheral parasitemia in the AZCQ group relative to SP suggest that AZCQ warrants further investigation as an alternative treatment of uncomplicated malaria. Trial Registration ClinicalTrials.gov (NCT01103063). This was a multi-center, Phase 3, open-label, randomized, clinical trial that compared the effectiveness of three IPTp courses of AZCQ or SP in pregnant women in five countries in sub-Saharan Africa where antifolate resistance of P. falciparum to SP was established. Women of all gravidities were enrolled during the second trimester of pregnancy and allocated to receive three IPTp courses of AZCQ or SP during ANC visits at 4- to 8-week intervals. They were then followed up at week 36 to 38 of gestation, at delivery (or within 2 days of study participant reporting home delivery), and on day 28 post delivery (time window: day 28 to 42). As the study was conducted in areas where P. falciparum resistance to SP was documented to reduce the protective efficacy of IPTp-SP [8,13,22–24], a superiority design was chosen. A composite primary endpoint was chosen so that all possible pregnancy outcomes that are potentially affected by uncontrolled parasitemia would be included in the analysis. The rationale for selection of endpoints and design features has been described in detail elsewhere [25]. The study was approved by the London School of Hygiene and Tropical Medicine Ethics Committee; the Comité National d’Ethique pour la Recherche en Santé in Cotonou, Benin; the Kenyatta National Hospital—University of Nairobi Ethics Review Committee in Nairobi, Kenya; the College of Medicine Research and Ethics Committee in Blantyre, Malawi; the Medical Research Coordinating Committee in Dar es Salaam, Tanzania; and the Uganda National Council of Science and Technology, the School of Medicine Research and Ethics Committee of Makerere University, and the Mulago Hospital Research and Ethics Committee in Kampala, Uganda. The study was overseen by an independent External Data Monitoring Committee (EDMC) and conducted in accordance with the Declaration of Helsinki on Ethical Principles for Medical Research Involving Human Study Participants; the International Conference on Harmonisation-Good Clinical Practice (ICH-GCP) standards; and local regulatory and legal requirements. Participants (or a legally acceptable representative if the participant was <18 years of age) were to provide written informed consent before enrollment and any study procedures took place. All study participants <18 years of age were to provide assent. The study was conducted between October 2010 and November 2013 at six sites: (1) the Centre de Sante d'Ahouansori Agué and Hôpital Bethesda in Cotonou, Benin; (2) the Siaya District Hospital, in Siaya, Kenya; (3) the Zomba Central Hospital in Zomba, Malawi; (4) The Teule Hospital in Muheza, Tanga, Tanzania; (5) the National Institute for Medical Research (Mwanza Centre)/Nyamagana District Hospital, in Mwanza, Tanzania; and (6) the Mulanda Health Centre IV, in Kampala, Uganda. Pregnant women of all gravidities were eligible if they carried a single fetus of 14 to 26 weeks of gestation (defined by pelvic ultrasound examination at screening), were 16 to 35 years of age, and willing and able to comply with all study procedures and to attend all scheduled follow-up visits. Women presenting at enrollment with clinical symptoms of malaria, severe anemia (hemoglobin <8 g/dL), any condition requiring hospitalization, obstetric complications increasing the risk of sub-optimal pregnancy outcome (e.g., presence of congenital anomalies, placenta previa, or abruption), evidence of severe concomitant infection, or who had taken antimalarial drugs within the past 4 weeks were excluded from enrollment (S1 Table). Study participants were randomly assigned (1:1) to the IPTp-AZCQ or the IPTp-SP regimen using computer-generated randomization cards provided by the sponsor to the investigators. Treatment group assignment remained concealed until the investigator confirmed the study participant met all eligibility criteria. Randomization was stratified according to gravidity into two approximately equal-sized strata (‘primi- and secundigravidae’ and ‘other gravidae’). Participants in both regimens received three IPTp courses: the first course between 14 and 26 weeks of gestation and the subsequent courses at 4- to 8-week intervals, with the third course administered prior to or during the 36th week of gestation. In addition, all participants also received a LLIN on day 0 of the study, and the installation of these nets was verified by fieldworkers on day 1 (AZCQ regimen) or day 2 (SP regimen). For the AZCQ combination, we used a fixed-dose tablet formulation of AZCQ 250/155 mg [26]; each IPTp treatment course consisted of a 3-day course of AZCQ 1,000/620 mg per day administered orally once daily on days 0, 1, and 2. For the SP regimen, we used fixed-dose tablets of sulfadoxine 500 mg plus pyrimethamine 25 mg supplied as Fansidar® (Roche); each treatment course consisted of a single dose of sulfadoxine 1,500 mg plus pyrimethamine 75 mg administered orally once on day 0. All study drug doses were administered under direct observation as open-label therapy. Administration of all SP doses and of the first dose of each 3-day AZCQ course was supervised by the investigators during ANC visits, and the second and third doses of each AZCQ treatment course were taken at home under supervision by fieldworkers. The composite primary efficacy endpoint was defined as the proportion of participants with a sub-optimal pregnancy outcome comprising: live-born neonate with LBW (defined as <2,500 g), premature birth (delivery before 37 weeks of gestation), still birth (pregnancy loss after 28 weeks of gestation), abortion (pregnancy loss before completion of 28 weeks of gestation), loss to follow-up prior to termination of pregnancy or delivery, and missing birth weight. Key secondary endpoints included the incidences of: LBW for live-born neonates, sub-optimal pregnancy outcome when including neonatal death and congenital malformation in addition to the six outcomes constituting the primary endpoint, maternal anemia (defined as hemoglobin <11 g/dL) and severe maternal anemia (defined as hemoglobin <8 g/dL) at weeks 36 to 38 of gestation, placental parasitemia at delivery, and of placental malaria infection as detected by histology at delivery; as well as, the number of episodes of curable sexually transmitted infections (STIs) with Treponema pallidum, Neisseria gonorrhoeae, and Chlamydia trachomatis per study participant (based on clinical presentation at any time between the first IPTp dose and delivery or laboratory results for specimens collected at weeks 36 to 38 of gestation). Other secondary efficacy endpoints were the incidences of: premature birth, still birth, congenital abnormalities in neonates (detected at birth); perinatal or neonatal deaths; study participants requiring additional treatment for malaria during the study period following first IPTp dose (diagnosed based on clinical presentation or laboratory test results); peripheral parasitemia at weeks 36 to 38 of gestation and at delivery; cord blood parasitemia at delivery; STIs with T. pallidum, N. gonorrhoeae, or C. trachomatis following the first IPTp dose (diagnosed based on clinical presentation before, or on laboratory test results between 36 to 38 weeks of gestation); positive laboratory test results for C. trachomatis, N. gonorrhoeae, T. pallidum, Trichomonas vaginalis, and bacterial vaginosis at 36 to 38 weeks of gestation; ophthalmia neonatorum in the neonate; bacterial infections including pneumonia and other lower respiratory tract infections between the first IPTp dose and delivery; pre-eclampsia between week 20 and delivery (diagnosed based on high blood pressure [systolic ≥140 mmHg or diastolic ≥90 mmHg in two separate measurements taken ≥4 hours apart] and proteinuria [≥300 mg protein in 24-hour urine collection]); and of nasopharyngeal swabs positive for macrolide-resistant and penicillin-resistant Streptococcus pneumoniae at baseline, at day 28 post delivery, and about 6 months following the last IPTp course (tested in ~600 study participants per treatment group); as well as the hemoglobin concentration at weeks 36 to 38 of gestation; the birth weight of live-born neonates; and the number of episodes of symptomatic malaria per study participant between the first IPTp dose and delivery. Safety endpoints included observed and spontaneously reported adverse events; vital signs; physical examination; laboratory tests (including tests for anemia, glycosuria, and proteinuria); routine obstetric checkup; adverse pregnancy outcomes for mothers; and the general physical examination of neonates through day 28 post delivery (time window: day 28 to 42). The training of investigators, site teams, and central laboratories as well as compliance and study monitoring have been described previously [25]. The study visits and procedures are outlined in S1 Fig. Participants with clinical episodes of malaria (defined as fever [oral temperature >37.5°C] and confirmation of malaria by rapid diagnostic test or light microscopy) received standard antimalarial treatment according to the local care guidelines, and continued follow-up. Study participants diagnosed with anemia received standard treatment according to local ANC guidelines. To investigate whether azithromycin exposure induced emergence of macrolide-resistant pneumococci, nasopharyngeal swabs were collected from a subset of participants (target: about 600 study participants per treatment arm) at baseline, day 28 post delivery, and 6 months after the last IPTp dose, and the sensitivity of isolated S. pneumonia to azithromycin, erythromycin, and penicillin was determined. Serious adverse events (SAEs) for mothers or neonates, which were observed or volunteered between signing of informed consent and day 28 to 42 post delivery, including 39 days after the last administration of investigational product, or the last study visit (whichever was later), were recorded and coded using the Medical Dictionary for Regulatory Activities (MedDRA, version 17.0), regardless of suspected causal relationship to study treatment. Adverse events (AEs) were recorded from the time the study participant had taken at least one dose of investigational product through to the last study visit. Patients were evaluated and questioned for AEs at each study visit. Severity and causality of AEs were assessed by the site investigator, with events considered ‘mild’, ‘moderate’, or ‘severe’ if there was no, some, or significant interference with the study participant’s usual function, respectively, and ‘treatment-related’ if there was a reasonable possibility that study treatment had contributed to or caused the event. AEs were classified as ‘serious’ if they were fatal or life-threatening; required inpatient hospitalization or prolongation of existing hospitalization; resulted in significant disability/incapacity; or were a congenital anomaly/birth defect. Every effort was made to document reasons for discontinuation and pregnancy outcomes for study participants who decided to withdraw from the study. If consent for disclosure of future information was also withdrawn, no further evaluations were performed; but all data collected up to the point of withdrawal remained in the database. In the event of safety concerns or failure to cooperate with study procedures, study participants were discontinued from study drug but not the study per se. All affected participants received standard ANC as per local guidelines, and were followed up regularly. Hemoglobin concentrations were quantified through finger prick or peripheral blood samples using HemoCueTM. The presence of peripheral, placental, and cord blood P. falciparum parasitemia was tested by microscopy using standard Giemsa-stained blood smears (thick and thin) at weeks 36 to 38 of gestation and at hospital delivery. Smears were read and, when positive, parasite density was counted independently by at least two microscopists at different laboratories, blinded to treatment regimen; discrepant results were reviewed by a third microscopist. The parasite count was expressed as the number of parasites per microliter of blood in a thick smear, standardized to a predetermined white cell count of 8000. A blood slide was considered negative when the examination of 100 high power fields on the thick smear did not show the presence of any falciparum parasites. In addition, if smears were microscopically-positive for P. falciparum at weeks 36 to 38 of gestation, the following P. falciparum genetic resistance markers were determined using polymerase chain reaction (PCR) assay: CQ resistance markers in the P. falciparum chloroquine resistance transporter (pfcrt) and multidrug resistance 1 (pfmdr1) genes, and SP resistance markers in the dihydrofolate (pfdhfr) and dihydropteroate synthase (pfdhps) genes. For placental histology, a sample of placenta tissue (approximately 2 cm x 2 cm x 1 cm) was collected at birth from the participants who delivered at hospital. Samples were placed in 50 mL 10% neutral buffered formalin, stored at room temperature, and shipped for histology review. The T. pallidum blood screening test was conducted using the Rapid Plasma Reagnin (RPR) method at baseline and at weeks 36 to 38. Blood samples (~0.5 ml) were collected and the serum used for the test. The Treponema Pallidum Particle Agglutination Assay (TPPA) was used to confirm infection when RPR was positive. N. gonorrhoeae and C. trachomatis tests were performed at weeks 36 to 38. An endocervical swab was collected and PCR assay (Amplicor CT/NG, Roche) used for analysis. All laboratory tests were standardized among units to enable comparison of test results from different laboratories. EAST software Version 5.1 and simulation were used to design the study and calculate the sample size required to detect a 20% risk reduction in the AZCQ treatment group relative to SP, for the primary endpoint of sub-optimal pregnancy outcome, with 90% power and a two-sided type I error rate (alpha) of 0.00125. The resulting sample size was also checked to ensure ≥80% power, at the 0.05 2-sided alpha level, for detecting a 23% risk reduction in the key secondary endpoint of LBW. The 2-sided significance level of 0.00125 (derived as 2 x 0.000625) for the primary endpoint equals the significance level of two independent confirmative trials each conducted at 0.05 2-sided alpha level. Note that to derive this: 0.025 is the 1-sided probability of a false positive for concluding superiority of AZCQ over SP in a single trial, with 0.025 x 0.025 = 0.000625 representing the same overall probability using two independent trials. The underlying true incidence of the composite primary endpoint in the control group was unknown at the design stage, but required for sample size considerations. So, the design planned one adaptive sample size refinement based on the observed pooled incidence of sub-optimal pregnancy outcomes, without regard to treatment regime, after collection of 1,000 observations for the primary endpoint. Based on the result from this adaptive sample size assessment, the sample size was refined to be 5,044 study participants (maximum allowed for per the protocol; S2 Text) if the study went to completion. The final protocol (S2 Text) included one interim analysis at 35% (i.e., following the completion of pregnancy outcome in the first 1,766 of 5,044 study participants to be randomized). Early termination for superiority was to occur if the interim analysis revealed a statistically significant lower risk of sub-optimal pregnancy outcome (primary endpoint) and of LBW (key secondary endpoint) in the AZCQ group compared with SP. The assessment of trial futility at the interim analysis (i.e., low likelihood that AZCQ will demonstrate benefit compared with SP in protective efficacy for IPTp should the study go to completion) was to be based solely on the primary endpoint of sub-optimal pregnancy outcome. Statistical stopping boundaries were employed based on controlling the overall study alpha at 0.00125 and 0.05 for the primary endpoint and LBW, respectively, to account for multiple queries of the data for these two endpoints. The EDMC reviewed interim analysis results and oversaw evaluation of emerging safety data on a regular basis. Although the study was open-label, study personnel involved in the day-to-day operation of the trial remained blinded to interim and aggregate treatment-group results until study termination. The primary analysis population was the intent-to-treat (ITT) population, including all study participants who were randomized, received at least one dose of study medication, and had a singleton gestation. Except for the primary endpoint, as defined above, there were no imputations for missing data. For dichotomous endpoints (including the primary endpoint), the percentage of study participants meeting the endpoint was estimated for each treatment arm and confidence intervals (CIs) were calculated using the normal approximation to the binomial. The risk ratio (RR) of the proportion of study participants meeting the endpoint (AZCQ/SP) was calculated to compare treatment groups. Mantel-Haenszel estimates of the common RR [27,28] stratified by randomization strata (gravidae) were computed utilizing the estimated variance given by Greenland and Robins [29], and two-sided p-values were calculated. Secondary endpoints involving counts, actual neonate birth weight, and hemoglobin values were analyzed using analysis models of variance (ANOVA) or of covariance (ANCOVA) if a baseline value was available. Model terms included treatment group, randomization strata, and, where applicable, the baseline value. Model adjusted means (least square [LS] means) and corresponding 95% CIs were computed for each treatment group and the difference between treatment groups (AZCQ minus SP). As noted above, stopping boundaries (i.e., alpha spending functions [30]) were employed to account for the interim analysis involving the primary endpoint and LBW. For the primary endpoint, the adjusted alpha level at the time of final study completion was 0.000109 for determining statistical significance. In the case of LBW, the adjusted alpha level at the final analysis was 0.003031. However, since the study was terminated early for futility based on the primary endpoint, all CIs for all endpoints were stated at the 95% confidence level for informational purposes only and all inferences for all secondary endpoints apart from LBW are to be considered exploratory. The safety populations included (i) all enrolled study participants who received at least one dose of study medication and (ii) all live-born babies. Descriptive statistics were used to summarize the data. Pfizer and the Medicines for Malaria Venture (MMV) funded this trial and were involved in study design, data analysis, data interpretation, and writing of the study report. All authors had full access to all the data in the study and had final responsibility for the decision to submit for publication. Medical writing and editorial support were provided by Susanne Vidot, PhD, of Engage Scientific and was funded by Pfizer.