Dihydroartemisinin-piperaquine for intermittent preventive treatment of malaria during pregnancy and risk of malaria in early childhood: A randomized controlled trial

Background: Intermittent preventive treatment of malaria in pregnancy (IPTp) with dihydroartemisinin-piperaquine (IPTp-DP) has been shown to reduce the burden of malaria during pregnancy compared to sulfadoxine-pyrimethamine (IPTp-SP). However, limited data exist on how IPTp regimens impact malaria risk during infancy. We conducted a double-blinded randomized controlled trial (RCT) to test the hypothesis that children born to mothers given IPTp-DP would have a lower incidence of malaria during infancy compared to children born to mothers who received IPTp-SP. Methods and findings: We compared malaria metrics among children in Tororo, Uganda, born to women randomized to IPTp-SP given every 8 weeks (SP8w, n = 100), IPTp-DP every 8 weeks (DP8w, n = 44), or IPTp-DP every 4 weeks (DP4w, n = 47). After birth, children were given chemoprevention with DP every 12 weeks from 8 weeks to 2 years of age. The primary outcome was incidence of malaria during the first 2 years of life. Secondary outcomes included time to malaria from birth and time to parasitemia following each dose of DP given during infancy. Results are reported after adjustment for clustering (twin gestation) and potential confounders (maternal age, gravidity, and maternal parasitemia status at enrolment).The study took place between June 2014 and May 2017. Compared to children whose mothers were randomized to IPTp-SP8w (0.24 episodes per person year [PPY]), the incidence of malaria was higher in children born to mothers who received IPTp-DP4w (0.42 episodes PPY, adjusted incidence rate ratio [aIRR] 1.92; 95% CI 1.00–3.65, p = 0.049) and nonsignificantly higher in children born to mothers who received IPT-DP8w (0.30 episodes PPY, aIRR 1.44; 95% CI 0.68–3.05, p = 0.34). However, these associations were modified by infant sex. Female children whose mothers were randomized to IPTp-DP4w had an apparently 4-fold higher incidence of malaria compared to female children whose mothers were randomized to IPTp-SP8w (0.65 versus 0.20 episodes PPY, aIRR 4.39, 95% CI 1.87–10.3, p = 0.001), but no significant association was observed in male children (0.20 versus 0.28 episodes PPY, aIRR 0.66, 95% CI 0.25–1.75, p = 0.42). Nonsignificant increases in malaria incidence were observed among female, but not male, children born to mothers who received DP8w versus SP8w. In exploratory analyses, levels of malaria-specific antibodies in cord blood were similar between IPTp groups and sex. However, female children whose mothers were randomized to IPTp-DP4w had lower mean piperaquine (PQ) levels during infancy compared to female children whose mothers received IPTp-SP8w (coef 0.81, 95% CI 0.65–1.00, p = 0.048) and male children whose mothers received IPTp-DP4w (coef 0.72, 95% CI 0.57–0.91, p = 0.006). There were no significant sex-specific differences in PQ levels among children whose mothers were randomized to IPTp-SP8w or IPTp-DP8w. The main limitations were small sample size and childhood provision of DP every 12 weeks in infancy. Conclusions: Contrary to our hypothesis, preventing malaria in pregnancy with IPTp-DP in the context of chemoprevention with DP during infancy does not lead to a reduced incidence of malaria in childhood; in this setting, it may be associated with an increased incidence of malaria in females. Future studies are needed to better understand the biological mechanisms of in utero drug exposure on drug metabolism and how this may affect the dosing of antimalarial drugs for treatment and prevention during infancy. Trial registration: ClinicalTrials.gov number NCT02163447. The study was funded by the National Institutes of Health and approved by the Institutional Review Boards of the Makerere University School of Biomedical Sciences, the Uganda National Council for Science and Technology, and the University of California, San Francisco. Written informed consent was obtained from all study participants. The study was conducted in Tororo district, Uganda, from June 2014 through May 2017. Tororo district is an area of historically high malaria transmission intensity with perennial transmission and an estimated entomologic inoculation rate of 310 infectious bites per person-year in 2013 [25]. Following a universal LLIN campaign in November 2013, near universal LLIN coverage was reported in Tororo district, with minimal change in malaria metrics after LLIN distribution [26]. From December 2014 to February 2015, indoor residual spraying (IRS) using the carbamate bendiocarb was initiated in Tororo district for the first time and was associated with significant reductions in malaria transmission [26,27]; a second round of bendiocarb was conducted in June–July 2015, and a third round in November–December 2015. A fourth round of IRS was conducted in June–July 2016 with pyrimiphos-methyl (Actellic), a long-lasting organophosphate. This study was divided into 2 phases, the first phase randomizing pregnant women to different IPTp regimens and the second phase—the focus of this analysis—following children born from these mothers to 2 years of age. In the first phase of this study, pregnant women were screened and enrolled between June 2014 and October 2014. Eligible mothers were not infected with HIV and were of all gravidities, with an estimated gestational age between 12 and 20 weeks, confirmed by ultrasound, and provided written informed consent. Complete entry criteria are provided (see S1 Study Protocol) and have been previously described [28]. In the second phase of this study, children were born between October 2014 and May 2015 and followed through 2 years of age, with the last participant followed through May 2017. This was a double-blinded RCT of pregnant women not infected with HIV and the children born to them. Women and their unborn child(ren) were randomized to one of five treatment arms, including both the intervention for the woman during pregnancy and her unborn child(ren) during infancy, in a 2:1:1:1:1 randomization scheme, as follows: (1) women IPTp-SP8w, children DP every 12 weeks; (2) women IPTp-DP8w, children DP every 12 weeks; (3) women IPTp-DP8w, children DP every 4 weeks; (4) women IPTp-DP4w, children DP every 12 weeks; and (5) women IPTp-DP4w, children DP every 4 weeks. To compare the malaria risk among infants whose mothers were randomized to different IPTp regimens, the prespecified protocol-defined study population included only mother/infant pairs in one of the three study arms randomized to receive DP every 12 weeks during infancy, because we hypothesized that children randomized to receive DP every 4 weeks in infancy would be nearly completely protected against malaria in infancy [29]. A randomization list using permuted blocks of 6 or 12 was computer generated by a member of the study not directly involved in patient care. Study participants were randomized to their assigned treatment group at enrolment using premade, consecutively numbered, sealed envelopes. Non-singleton births from the same mother were assigned to the same intervention. Study pharmacists not otherwise involved in the study were responsible for treatment allocation and the preparation of study drugs. In pregnancy, each treatment with SP (Kamsidar, Kampala Pharmaceutical Industries, 500/25 mg tablets) consisted of 3 tablets given as a single dose. Each treatment with DP in pregnancy (Duo-Cotexin, Holley-Cotec, Beijing, China, 40 mg/320 mg tablets) consisted of 3 tablets given once a day for 3 consecutive days. Participants allocated IPTp-SP8w or IPTp-DP8w received active study drugs at 20, 28, and 36 weeks gestational age. Participants allocated IPTp-DP4w received active study drugs starting at 16 or 20 weeks gestational age. Placebos of SP and DP were used such that every 4 weeks participants received the same number of pills, with the same appearance. Each treatment with DP in childhood consisted of half-strength tablets given once a day for 3 consecutive days (Duo-Cotexin, Holley-Cotec, Beijing, China, 20 mg/160 mg tablets), according to weight-based guidelines (see S1 Study Protocol). Infants randomized to receive DP every 12 weeks received placebo mimicking the dosing of DP every 4 weeks when they were not receiving study drug. The first day of each dose was directly observed in the clinic by study nurses, who had the flexibility to administer either intact or crushed tablets to the infants. Compliance with day 2 and day 3 dosing administered at home was assessed at the following monthly routine visit and was reported to be >99%. At enrolment, women received an LLIN and underwent a standardized examination. Pregnant women and their children received all of their medical care at a study clinic open every day. Study procedures for pregnant women have previously been described, with details available in S1 Study Protocol [8]. Briefly, during pregnancy, routine visits were conducted every 4 weeks, including collection of dried blood spots (DBS) for molecular testing, and women were encouraged to deliver at the hospital adjacent to the study clinic. At delivery, a standardized assessment was completed, including evaluation of birth weight and collection of biological specimens, including maternal blood, placental tissue, placental blood, and cord blood. Following delivery, children were followed through 24 months of age and encouraged to come to the study clinic any time they were ill. Those who presented with a documented fever (tympanic temperature ≥38.0 ˚C) or history of fever in the previous 24 hours had blood collected for a thick blood smear. If the smear was positive, the patient was diagnosed with malaria and treated with AL. If the thick blood smear was negative, the patient was managed for a non-malarial febrile illness by study physicians. Episodes of uncomplicated malaria in children <4 months of age or weighing <5 kg, as well as episodes of complicated malaria and treatment failures within 14 days, were treated with either quinine or artesunate according to national malaria treatment guidelines. Routine visits were conducted every 4 weeks in children, including thick blood smears to assess for parasitemia by microscopy, collection of DBS for molecular testing, and collection of plasma by fingerprick for assessment of piperaquine (PQ) levels. Phlebotomy for routine laboratory tests, including complete blood count (CBC), was performed every 16 weeks. Adverse events were assessed and graded according to standardized criteria at every visit to the study clinic [30]. Blood smears were stained with 2% Giemsa and read by experienced laboratory technologists. A blood smear was considered negative when the examination of 100 high-power fields did not reveal asexual parasites. For quality control, all slides were read by a second microscopist, and a third reviewer would settle any discrepant readings. DBS were tested for the presence of malaria parasites using a loop-mediated isothermal amplification (LAMP) kit (Eiken Chemical, Japan). IgG responses to 19 parasite surface antigens were measured in maternal and cord blood collected at delivery, including circumsporozoite protein (CSP), erythrocyte binding antigen (EBA) 140 region III-V (RIII-V), EBA175 RIII-V [31,32], EBA181 RIII-V, Early transcribed membrane protein (Etramp) 4, Etramp 5, gametocyte exported protein (GEXP18), H103/merozoite surface protein (MSP) 11, Heat shock protein 40 (HSP40), Plasmodium exported protein (Hyp2) [33]), MSP 2 (Ch150/9 and Dd2 alleles) [34], Apical membrane antigen 1 (AMA1) [31,32,35], glutamate rich protein (GLURP-R2)[36], MSP1-19 [31,32,37], Schizont egress antigen (SEA)-1 [38], Reticulocyte-binding protein homologue (Rh)2_2030, Rh4.2, and skeleton-binding protein 1 (SBP1). Glutathione S-transferase (GST) and Tetanus toxoid were used as controls. Luminex magnetic microsphere conjugation was performed by standard methods [39]. Fifty microliters thawed plasma (1/1,000 dilution) were coincubated with microsphere mixtures on a 96-well plate for 90 minutes, washed, then stained with 50 uL of 1/200 R-Phycoerythrin-conjugated AffiniPure F(ab’)2 Goat anti-human IgG (Jackson Immuno Research Laboratories) secondary antibody. Samples were then suspended in 100 uL PBS and read by the Luminex MAGPIX system. Positive control samples from individuals (n = 20) with known antibodies to these antigens were included on each plate. Standard curves were generated through serial dilutions of the positive control pool. Antibody levels, expressed in arbitrary units (AUs), were obtained by regressing raw MFI onto the standard curve for each antigen present on every plate and results log transformed [40]. Children provided capillary blood samples at 3 consecutive routine visits performed every 4 weeks after they received the 8, 32, 56, and 92 week doses of DP in infancy. Pharmacokinetic samples (n = 1,505) were centrifuged within 60 minutes at 2,000g for 10 minutes, and plasma was stored at −80°C prior to being processed for PQ quantitation. PQ concentrations were determined using high performance liquid chromatography tandem mass spectrometry, as described [41], with modifications to lower the calibration range to 0.5–50 ng/mL and a new calibration range of 10–1,000 ng/mL. The lower limit of quantification (LLOQ) was 0.5 ng/mL and the coefficient of variance was <10% for all quality control concentrations. The primary outcome was the incidence of malaria from birth to 24 months of age. Treatments for malaria within 14 days of a prior episode were not considered incident events. Secondary outcomes included time to malaria from birth and time to parasitemia following receipt of each dose of DP; the incidence of complicated malaria; the incidence of hospitalizations/deaths; the incidence of non-malarial febrile illness (presentation within 14 days of a prior episode were not considered incident events); and the prevalence of anemia (Hb < 11 g/dL) during infancy. Measures of safety included the incidence of adverse events from birth through 2 years of age. Post hoc, exploratory outcomes included the relative intensity of malarial antibodies measured at delivery (maternal and cord); and PQ levels measured 4, 8, and 12 weeks following receipt of DP. The primary exposure variable was maternal IPTp assignment. To test the hypothesis that either IPTp-DP4w or IPTP-DP8w would be associated with a lower risk of malaria in infancy compared to SP, we assumed an incidence of malaria of 3–5 episodes per person year (PPY) among children whose mothers were randomized to IPTp-SP8w based on prior data before the implementation of IRS. Assuming 5% lost to follow-up, we had 80% power to show a 22%–28% reduction in the incidence of malaria among infants whose mothers were randomized to either IPTp-DP4w or IPTp-DP8w (2-sided significance level = 0.05). Data were double-entered and verified in Microsoft Access and statistical analysis performed using Stata, version 14. All analyses were done using a modified intention-to-treat approach, including all children born (excluding stillbirths) and randomized to DP every 12 weeks with evaluable person-time of follow-up. Any premature withdrawal from the study prior to 2 years of age was assumed to be random. Comparisons of simple proportions were made using the chi-squared or Fisher’s exact test. Comparisons of incidence measures were made using a negative binomial regression model. We assessed for significant interaction (p < 0.10) with the primary outcome and the following potential effect modifiers: sex of the infant, gestational age of the infant at birth, and maternal gravidity. Where significant effect modification was noted, results were reported from stratified analysis. The cumulative risk of developing malaria from birth was estimated using the Kaplan–Meier product limit formula, and associations with exposure variables assessed using a cox proportional hazards model. The cumulative risk of developing malaria parasitemia following receipt of each dose of DP in infancy was estimated using a multilevel mixed-effects parametric survival model, accounting for clustering within individuals and mothers (twin gestation). Comparisons of proportions with repeated measures were made using mixed effects logistic regression models. In all analyses, estimates accounted for maternal clustering (twin gestation). Estimates were adjusted for potential confounders (maternal age, gravidity, and maternal parasitemia status at enrolment); both unadjusted and adjusted results were reported in tables; adjusted estimates are presented in the text. In post hoc analyses, comparisons of log-transformed antibody levels between groups were performed using the student t test. For PQ measurements, relationships between mean population PQ concentrations, days since dosing, maternal randomization, and infant sex were assessed using generalized estimating equations with log link and robust standard errors accounting for repeated observations within individuals. Marginal estimates were produced using final models and shown graphically. In all analyses, p < 0.05 was considered statistically significant, without adjustment for multiple comparisons.