Ethics statement

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.

Study setting and 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.

Study design and randomization

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.

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%.

Study procedures

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].

Clinical laboratory procedures

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).

Antibody measurements

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].

PQ pharmacokinetic sample collection and quantitation

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.

Study end points

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.

Statistical analysis

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.

Study participants and follow-up

Between June 2014 and October 2014, 386 pregnant women not infected with HIV and their unborn children were screened, and 300 were enrolled and randomized to one of five treatment arms defined by both maternal IPTp (SP8w, DP8w, or DP4w) and infant intermittent preventive treatment (IPT) (DP every 4 or 12 weeks beginning at 8 weeks of age, Fig 1). Among pregnant women/child pairs enrolled, 202 were randomized to receive DP every 12 weeks in infancy and included in this analysis. Of these, 106 women were randomized to IPTp-SP8w; 46 to IPTp-DP8w, and 50 to IPTp-DP4w. A total of 192 women (95.0%) were followed through delivery, and 4 women gave birth to twins. Of 194 live births, 191 children were followed for at least one day and included in the analyses. All children were born between October 2014 and May 2015.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 1. Trial profile.

DP, dihydroartemisinin-piperaquine; IPTp-DP4w, IPTp-DP every 4 weeks; IPTp-DP8w, IPTp-DP given every 8 weeks; IPTp-SP8w, IPTp-SP given every 8 weeks.

https://doi.org/10.1371/journal.pmed.1002606.g001

Mean maternal age at enrolment was 22 years, 69% of women were enrolled at ≤16 weeks gestational age, 37% were primigravida, and 57% had malaria parasites detected by LAMP. Baseline maternal characteristics were not significantly different between the 3 IPTp arms, except for age, with women randomized to IPTp-SP8w slightly younger than women randomized to the other 2 treatment groups (Table 1). After initiation of IPTp, the incidence of malaria was significantly higher during pregnancy in mothers who received IPTp-SP8w (0.95 episodes PPY) compared to mothers who received IPTp-DP8w (0.23 episodes PPY, p = 0.004) or IPTp-DP4w (0 episodes PPY, p < 0.001). Similarly, parasite prevalence during pregnancy was significantly higher in mothers who received IPTp-SP8w compared to mothers who received IPTp-DP8w or IPTp-DP4w. The prevalence of placental malaria determined by LAMP or histopathology was significantly higher among women who received IPTp-SP8w compared to women who received IPTp with either IPTp-DP8w or IPTp-DP4w (Table 1).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 1. Characteristics of mother-child pairs stratified by mother’s IPTp treatment arm.

https://doi.org/10.1371/journal.pmed.1002606.t001

Of 191 children born and followed, 98 (51.3%) were female, 14 (7.3%) were born preterm, and 21 (11%) were born with LBW (<2,500 g), and these characteristics were not significantly different based on maternal IPTp. Median gestation age at birth was slightly older for babies born to mothers who received IPTp-DP4w (median gestation age 40 weeks [35.7–41.6 weeks]) compared to those born to mothers who children whose mothers received IPTp-SP8w (median gestation age 39.3 weeks [31.7–42.0 weeks], p = 0.03, Table 1). Of 191 children followed, 164 (86%) completed follow-up through 2 years of age (Fig 1). When considering person-time of follow-up, 355.1 of 382 (93%) possible person years were observed.

Impact of IPTp with DP on burden of malaria in infancy

During 355.1 person years of follow-up, there were 105 incident episodes of malaria in infants, giving an overall incidence of 0.30 episodes PPY. Compared to children born to mothers who received IPTp-SP8w, the incidence of malaria during 24 months of follow-up was higher in children born to mothers who received IPTp-DP4w (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 (aIRR 1.44; 95% CI, 0.68–3.05; p = 0.34, Table 2). However, we observed significant effect modification by infant sex. Female children born to mothers who received IPTp-DP4w had a greater than 4-fold apparently higher incidence of malaria (aIRR 4.39; 95% CI, 1.87–10.3; p = 0.001) compared to female children born to mothers who received IPTp-SP8w. Similarly, female children born to mothers who received IPTp-DP8w had a higher, but nonsignificant, risk of malaria compared to female children born to mothers who received IPTp-SP8w (aIRR 2.43; 95% CI, 0.85–6.97; p = 0.10). In contrast, there were no significant differences in the incidence of malaria among male children across the 3 IPTp treatment arms (Table 2). No significant effect modification was observed with maternal gravidity or gestational age at birth.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 2. Incidence of malaria though 24 months of age by mother’s IPTp treatment arm overall and stratified by infant sex.

https://doi.org/10.1371/journal.pmed.1002606.t002

Children born to mothers who received IPTp-DP4w had a nonsignificantly increased hazard of developing a first episode of malaria during the first 2 years of life in comparison to children born to mothers who received IPTp-SP8w (adjusted hazard ratio [aHR] 1.76, 95% CI 0.93–3.35, p = 0.08), but we again observed significant interaction with infant sex. Female infants whose mothers received IPTp-DP4w had a 3-fold apparently increased hazard of developing malaria compared to infants born to mothers who received IPTp-SP8w (aHR 3.40; 95% CI,1.55–7.96; p = 0.002, Table 3). Female children born to mothers who received IPTp-DP8w also had a higher, but nonsignificant, increased risk of malaria compared to those whose mothers received IPTp-SP8w; no significant associations were observed among male children across the 3 IPTp treatment arms (Table 3). These data do not support our original hypothesis that preventing malaria exposure in utero with DP leads to a reduced incidence of malaria in childhood; in contrast, in this setting it may be associated with an increased incidence of malaria in females.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 3. Time to first episode of malaria by mother’s IPTp treatment arm overall and stratified by infant sex.

https://doi.org/10.1371/journal.pmed.1002606.t003

Given these differences, we assessed whether the incidence of malaria was different between male and female children. Overall, female children had a nonsignificantly increased incidence of malaria compared to males (aIRR 1.43, 95% CI 0.82–2.50, p = 0.21), but this effect was modified by maternal IPTp. Female children whose mothers were randomized to IPTp-DP4w had a >3-fold apparently increased risk of malaria compared to males (aIRR 3.34, 95% CI 1.08–10.26, p = 0.04). However, there were no significant sex-specific differences among children whose mothers were randomized to IPTp-SP8w (aIRR 0.72, 95% CI 0.31–1.67 comparing females to males) or IPTp-DP8w (aIRR 1.66, 95% CI 0.59–4.67 comparing females to males). These data suggest that, in this setting, female infants appear to have an increased risk of malaria in infancy compared to males, but only among those whose mothers received IPTp-DP4w.

After observing an apparently higher risk of clinical malaria among female infants whose mothers received IPTp-DP4w compared to those whose mothers received IPTp-SP8w, we next sought to determine whether these infants were at an increased risk of P. falciparum infection in infancy versus an increased risk of symptoms if infected. As children followed in this study received curative IPT with DP every 12 weeks beginning at 8 weeks of age, and had both passive and active surveillance for P. falciparum infection, we assessed time to parasitemia following each dose of DP in infancy. Overall, children born to mothers who received IPTp-DP4w had an increased hazard of parasitemia following receipt of DP in infancy compared to children born to mothers who received IPTp-SP8w (aHR 2.02, 95% CI 1.08–3.79, p = 0.03, Fig 2A, Table 4), but we again observed significant interaction with infant sex. Female infants whose mothers had received IPTp-DP4w had a more than 5-fold apparently increased hazard of developing parasitemia following receipt of DP in infancy than children born to mothers who received IPTp-SP8w (aHR, 5.02; 95% CI, 1.90–13.2, p = 0.001, Fig 2B, Table 4). There was no significant difference in the hazard of developing parasitemia among male children born to mothers who received IPTp-DP4w or IPTp-DP8w compared to male children born to mothers who received IPTp-SP8w (Table 4, Fig 2C). Prior to first receipt of DP at 8 weeks of age, parasitemia was rare: 1/182 (0.55%) were LAMP positive at week 4, and 3/183 (1.64%) were microscopy or LAMP positive at 8 weeks of age. All 4 children with parasitemia at or before 8 weeks of age were born to mothers who received IPTp-SP8w, and none of these episodes were symptomatic. These data suggest that the increased incidence of malaria among female infants whose mothers received IPTp-DP4w is directly related to an increased risk of blood stage P. falciparum infection following receipt of DP in infancy.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 2. Time to parasitemia following DP administration in infancy.

Repeated measures analysis performed, using multilevel mixed-effects survival model, account for clustering within individuals and mothers, both overall (A) and stratified by infant sex (B–C) and adjusted for maternal age, gravidity, and LAMP status at enrolment. *aHR and p-values comparing IPTp-DP4w versus IPTp-SP8w. aHR, adjusted hazard ratio; DP, dihydroartemisinin-piperaquine; IPTp, intermittent preventive treatment of malaria in pregnancy; IPTp-DP4w, IPTp-DP every 4 weeks; IPTp-DP8w, IPTp-DP every 8 weeks; IPTp-SP8w, IPTp-SP given every 8 weeks; LAMP, loop-mediated isothermal amplification.

https://doi.org/10.1371/journal.pmed.1002606.g002

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 4. Time to parasitemia following each dose of DP by mother’s IPTp treatment arm overall and stratified by infant sex.

https://doi.org/10.1371/journal.pmed.1002606.t004

Secondary clinical and safety outcomes

During 2 years of follow-up, there were 9 episodes of complicated malaria: 4 among the 100 infants whose mothers received IPTp-SP8w (0.02 episodes PPY), 2 among 44 infants whose mothers received IPTp-DP8w (0.02 episodes PPY), and 3 among 47 infants whose mothers received IPTp-DP4w (0.04 episodes PPY). The risk of complicated malaria did not significantly differ between IPTp groups, nor between infant sex (5 episodes in males, 4 in females). Of the 9 episodes, 8 occurred between 12 and 24 months of age, 6 of 9 represented the child’s first episode of malaria and were associated with 1–2 convulsive episodes, and no episodes of cerebral malaria or severe anemia were observed. There were 3 deaths among infants included in this study. None of the deaths were due to malaria. Overall, there were 18 hospitalizations or deaths, with no significant difference in the number of hospitalization or deaths between IPTp treatment arms (Table 5). Children whose mothers received IPTp-DP4w or IPTp-DP8w had a nonsignificantly lower risk of anemia (hemoglobin <11 g/dL) compared to children whose mothers received IPTp-SP8w. Children born to mothers who received IPTp-DP4w or IPTp-DP8w also had a nonsignificantly lower incidence of non-malarial febrile illnesses compared to children born to mothers who received IPTp-SP8w (Table 5). These results are consistent with one other observational birth cohort, which found that malaria in pregnancy was associated with a higher risk of non-malarial febrile illness [42]. There were no significant differences in the incidence of adverse events of any severity, grade 3–4 adverse events, or serious adverse events (S1 Table). No adverse events were thought to be related to study drugs.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Table 5. Secondary outcomes by mother’s IPTp regimen.

https://doi.org/10.1371/journal.pmed.1002606.t005

Impact of IPTp with DP on antibody responses in infancy

Given the apparent sex-specific differences in malaria risk observed among infants whose mothers were randomized to different IPTp regimens, we then performed exploratory analyses to evaluate potential mechanistic explanations. As passive transfer of protective antibodies from the mother to the newborn may lead to a reduced risk of malaria early in life [37,43], we first assessed the impact of maternal IPTp regimens on malaria-specific IgG antibody levels in maternal and infant cord blood using a high-sensitivity Luminex-based, multiplex fluorescent bead-based assay. Although antibody levels to several antigens were significantly lower at delivery among mothers who received IPTp-DP4w in comparison to mothers who received IPTp-SP, IgG antibody levels measured in infant cord blood were similar between groups for all antigens (Fig 3). Furthermore, no significant sex-specific differences in antibody levels were observed. Therefore, the increased malaria risk observed among female infants whose mothers received IPTp-DP4w did not appear to be due to decreased malaria-specific antibody levels secondary to suppression of infection.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 3.

Antibody levels stratified by maternal IPTp assignment (IPTp-SP8w versus IPTp-DP4w) measured in maternal blood at delivery (A) and infant cord blood (B). *p < 0.05; **p < 0.01. AMA1, Apical membrane antigen 1; CSP, circumsporozoite protein; EBA, erythrocyte binding antigen; Etramp4, Early transcribed membrane protein 4; GEXP, gametocyte export protein; GST, Glutathione S-transferase; H103/MSP11, merozoite surface protein 11; HSP40, Heat shock protein 40; Hyp2, Plasmodium exported protein; IPTp, intermittent preventive treatment of malaria in pregnancy; IPTp-DP4w, IPTp-DP every 4 weeks; IPTp-SP8w; IPTp-SP every 8 weeks; MSP1, merozoite surface protein 1; MSP2^Ch150 and MSP2^Dd2, merozoite surface protein 2 of Ch150/90 and Dd2 alleles; Rh2, Reticulocyte-binding protein homologue 2; Rh4, Reticulocyte-binding protein homologue 4; SBP1, skeleton-binding protein 1; SEA, Schizont egress antigen; TT, tetanus toxoid.

https://doi.org/10.1371/journal.pmed.1002606.g003

Exposure to DP during pregnancy and PQ levels in infancy

Because PQ trough levels are strongly predictive of malaria risk among children receiving IPT with DP [29], we next assessed whether there were sex-specific differences in PQ levels in infancy and whether in utero exposure to DP alters these PQ levels. Capillary PQ levels were measured 28, 56, and 84 days post the 8, 32, 56, and 92 week doses of DP in infancy (n = 1,505 measurements). Female children whose mothers were randomized to IPTp-DP4w had 19% lower mean 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). In contrast, male children whose mothers were randomized to IPTp-DP4w had 17% higher mean PQ levels during infancy compared to males whose mothers received IPTp-SP8w, although not significantly so (coef 1.17, 95% CI 0.92–1.50, p = 0.21). In addition, female children whose mothers were randomized to IPTp-DP4w had 28% lower mean PQ levels post-dose than male children (coef 0.72, 95% CI 0.57–0.91, p = 0.006, Fig 4C). However, there were no significant sex-specific differences among children whose mothers were randomized to IPTp-SP8w (coef 1.07, 95% CI 0.86–1.32, p = 0.57, Fig 4A) or IPTp-DP8w (coef 1.09, 95% CI 0.97–1.38, p = 0.44, Fig 4B). Together, these data suggest that consistent in utero exposure to DP may lead to sex-specific differences in PQ metabolism in infancy, with lower PQ levels following administration among female infants.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Fig 4.

Mean PQ levels measured 28, 56, and 84 days posttreatment among children whose mothers were randomized to IPTp-SP8w (A), IPTp-DP8w (B), and IPTp-DP4w (C). Marginal estimates obtained using generalized estimating observations with log link and robust standard errors, accounting for repeated measurements in children. *p < 0.05. **p < 0.01. DP, dihydroartemisinin-piperaquine; IPTp, intermittent preventive treatment of malaria in pregnancy; DP4w, IPTp-DP given every 4 weeks; DP8w, IPTp-DP given every 8 weeks; SP8w, IPTp-SP given every 8 weeks; PQ, piperaquine; SP, sulfadoxine-pyrimethamine.

https://doi.org/10.1371/journal.pmed.1002606.g004