Abstract

Background. Factors involved in the development of resistance to sulphadoxine-pyrimethamine (SP) by Plasmodium falciparum, particularly in the context of intermittent preventive treatment during pregnancy (IPTp), are not well known. We aimed to determine the impact of IPTp and human immunodeficiency virus (HIV) infection on molecular markers of SP resistance and the clinical relevance of resistant infections.

Methods. SP resistance alleles were determined in peripheral (n = 125) and placental (n = 145) P. falciparum isolates obtained from pregnant women enrolled in a randomized, placebo-controlled trial of IPTp in Manhiça, Mozambique.

Results. Prevalence of quintuple mutant infections was 12% (23 of 185 isolates) in pregnant women who received placebo and 24% (20 of 85 isolates) in those who received SP (P = .031). When the last IPTp dose was administered at late pregnancy, mutant infections at delivery were more prevalent in placental samples (7 [23%] of 30, samples) than in peripheral blood samples (2 [7%] of 30 samples; P = .025), more prevalent in women who received IPTp-SP than in those who received placebo (odds ratio [OR], 8.13; 95% confidence interval [CI], 1.69–39.08), and more prevalent in HIV-positive women than in HIV-negative women (OR, 5.17; 95% CI, 1.23–21.66). No association was found between mutant infections and increased parasite density or malaria-related morbidity in mothers and children.

Conclusions. IPTp with SP increases the prevalence of resistance markers in the placenta and in HIV-infected women at delivery, which suggests that host immunity is key for the clearance of drug-resistant infections. However, this effect of IPTp is limited to the period when blood levels of SP are likely to be significant and does not translate into more-severe infections or adverse clinical outcomes.

Prompt diagnosis and effective treatment of malaria episodes are recommended for the control of malaria throughout the tropics. However, the success of Plasmodium falciparum treatment with antimalarials is compromised by drug resistance and the lack of alternative drugs. The threat of antimalarial resistance is even greater for pregnant women because of their increased risk of malaria [1] and the limited availability of antimalarial drugs that have been proven safe and effective in pregnancy [2]. Moreover, pregnancy entails physiological changes that can alter drug disposition and metabolism [3], particularly in the placenta, where parasites accumulate to high densities [4]. Also, conditions known to impair maternal immunity, such as human immunodeficiency virus (HIV) co-infection [5], may alter the efficacy of antimalarials, which depends not only on the susceptibility of the parasite but also, importantly, on host immunity [6].

Although sulphadoxine-pyrimethamine (SP) is not currently used for first-line malaria treatment because of its low therapeutic efficacy, it is provided to pregnant women in most African countries as intermittent preventive treatment (IPTp). This intervention has shown considerable benefits to the mother and the fetus [7, 8]. However, the use of slowly eliminated SP [9] inherently risks increasing drug resistance, which is acquired through the stepwise accumulation of point mutations in the parasite genes encoding the dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) enzymes of the folate metabolism [10]. Quintuple mutations in dhfr-51, -59, and -108 and dhps-437 and -540 codons confer high levels of drug resistance in vitro [11]. Although chemoprophylaxis of malaria in pregnancy with pyrimethamine has been associated with the selection of dhfr triple mutations in the placenta [12], self-reported use of IPTp at the same study site [13] did not increase the prevalence of resistant alleles. Observational studies in other settings support this finding [14]. In contrast, a recent study showed an increased fraction of mutant parasites among women who self-reported IPTp use [15]. Moreover, contradictory results about the impact of mutant infections on pregnancy outcomes have been reported [12, 15].

To understand the potential contribution of mutated parasites to malaria-related morbidity in mothers and children and the mechanisms underlying the emergence of drug resistance, we aimed to assess the impact of IPTp and maternal HIV infection on the prevalence of molecular markers of SP resistance. To address this, we analyzed P. falciparum isolates collected at delivery from Mozambican pregnant women participating in a randomized, double-blind, placebo-controlled trial of IPTp with SP.

MATERIAL AND METHODS

Study Area and Population

The study was conducted at the Centro de Investigação em Saúde da Manhiça (CISM) in Manhiça District of southern Mozambique during the period 2003–2005, before IPTp-SP was recommended by the Ministry of Health. In this area, perennial malaria transmission with some seasonality is mostly attributable to P. falciparum [16], and the estimated entomological inoculation rate for 2002 was 38 infective bites per person per year. During the study, malaria control in pregnancy relied exclusively on case management with chloroquine (CQ) plus SP for cases of uncomplicated malaria (in the first trimester CQ alone was given). At that time, highly active antiretroviral therapy was not available, and national policy on prevention of mother-to-child transmission of HIV infection was self-administration of nevirapine to the mother at the onset of labor and to the newborn within the first 72 h of life [17].

Study Design

This study was done in the context of a randomized, double-blind, placebo-controlled trial of IPTp with SP (trial registration number: NCT00209781) [17]. Pregnant women were enrolled into the study if they agreed to participate and signed the informed consent form. If their gestational age was ≤28 weeks, pregnant women received a long-lasting insecticide–treated net and were randomized to receive SP or placebo twice from the second trimester, at least 1 month apart. At delivery, birth weight was measured and blood samples were collected from the placenta, cord, and periphery of the mothers for hematological and parasitological examination. Impression smear and placental biopsy specimens were collected from the maternal side of the placentas for histological examination. Peripheral and placental blood samples were also collected onto filter papers (Schleicher & Schuell number 903TM). Hematocrit levels of the mothers and their children were measured 1 month after delivery, and clinical malaria episodes in women until 8 weeks postpartum and in infants during the first year of life were recorded through passive detection of cases reported at the Manhiça outpatient clinic.

Filter papers corresponding to blood samples from pregnant women of known HIV status and a peripheral microscopic P. falciparum infection (n = 86) or a histological-confirmed placental active infection (defined as the presence of parasites alone or together with pigment; n = 104) were included in the study. Also, an HIV-stratified random selection of 350 paired peripheral-placental blood samples from slide-negative pregnant women was included for the analysis of submicroscopic infections. The study was approved by the national Mozambican ethics review committee and the Hospital Clínic of Barcelona ethics review committee.

Laboratory Methods

Thin and thick films, impression smears, and placental biopsy specimens were read according to standard, quality-controlled procedures [18, 19]. Hematocrit levels were measured in a microcapillary tube after centrifugation. Maternal HIV type 1 status was determined with the Determine HIV-1/2 Rapid Test (Abbott Laboratories) and was confirmed with the Unigold rapid test (Trinity Biotech) [17].

DNA was extracted from a drop of 50 μL of blood onto filter paper with an ABIPrism 6700 Automated Nucleic Acid Work Station (Applied Biosystems) and resuspended into a final volume of 200 μL of water. Five microliters of the DNA extracts were screened for P. falciparum DNA by real-time quantitative polymerase chain reaction (qPCR) [20]. Parasitemia was quantified using the ABI Prism SDS2.1 software by extrapolation of cycle thresholds from a standard curve of known numbers of ring-infected erythrocytes diluted in whole blood. A negative control sample with no template DNA was also run in all reactions.

P. falciparum dhfr-51, -59, and -108 and dhps-437 and -540 genotypes were determined by restriction fragment-length polymorphism analysis of amplicons generated by nested PCR and electrophoresis on agarose gels [21]. Multiplicity of infection (MOI) was determined as the highest number of msp-1 or msp-2 PCR products detected in the sample [22]. Molecular analysis was blinded to origin of the sample (placental or peripheral), treatment group allocation (SP or placebo), and HIV status.

Definitions and Statistical Analysis

Infections were defined as pure quintuple mutants (q-mutants) if all 5 dhfr/dhps mutations were detected and there was no evidence of any wild-type parasite. Pregnant women were classified into first-time mothers (primigravidae; PG) and those with at least 1 previous pregnancy (multigravidae; MG). Age was stratified as ≤20, 21–25 and >25 years. Placental inflammation was defined by >5 inflammatory cells seen by histological examination in 10 high-power fields (original magnification, ×400). Late IPTp treatment was defined when pregnant women received the last SP dose within 2.5 months before delivery (median period of time between last SP dose and delivery among the women participating in the study; interquartile range, 1.9–3.4 months) and early-IPTp treatment if the interval between last SP dose and delivery was >2.5 months. Statistical analysis was performed using Stata software, version 11.0 (StataCorp). Proportions and log-transformed parasite densities were compared by Fisher's exact test and Student's t test, respectively. McNemar's test was used to compare prevalences of q-mutants among paired placental and peripheral samples. Logistic regression models were estimated to evaluate the association of q-mutants with early and late-IPTp, placental inflammation, parity, age, and HIV infection. Linear models were used to estimate the association of q-mutants with log-transformed parasite densities, MOI, newborn weight, and hematocrit levels in mothers, cords, and infants. Poisson regression models were used to evaluate the association of q-mutants with the number of malaria episodes in mothers during the postpartum period and in their children during the first year of life. Univariate and multivariate models adjusted for HIV infection, parity, and age were estimated. Two-tailed P values <.05 were considered statistically significant.

RESULTS

The study comprised 890 blood filter papers obtained at delivery from 454 placentas (104 with evidence of active P. falciparum infection on histological examination and 62 with evidence of submicroscopic infections) and 436 peripheral blood samples (86 of which were positive for P. falciparum on microscopic examination and 73 of which showed evidence of submicroscopic infections) of pregnant women enrolled in the IPTp trial [17].

Among the 325 P. falciparum–positive samples, genotypes for msp-1/msp-2 and the 5 dhfr/dhps codons were finally obtained for 270 isolates (83%) (145 from placental blood specimens and 125 from peripheral blood specimens; Table 1) from 171 pregnant women, including 99 with matched placental-peripheral blood infections, 46 with only placental infection, and 26 with only peripheral blood infection. Maternal age, parity, and HIV status were comparable between treatment groups (Table 1). No statistically significant difference was found by intervention group in microscopic parasite densities per microliter in peripheral blood (placebo group [n = 38]: mean density ± standard deviation [SD], 2726.8 ± 5920.9; SP group [n = 22]: mean density ± SD, 6508.6 ± 13415.3; P = .133) or in placental blood (placebo group [n = 43]: mean density ± SD, 5697.5 ± 12591.8; SP group [n = 21]: mean density ± SD, 11481.6 ± 23862.9; P = .229). However, qPCR densities were significantly lower in SP recipients than in placebo recipients (P = .041 for peripheral blood infection and <.001 for placental blood infections; Table 1). MOI of placental and peripheral blood infections was also lower in women who received SP than in those who received placebo, although the differences were not statistically significant (Table 1).

Table 1.

Baseline Characteristics of Pregnant Women included in the Study

 Women with peripheral blood infection (n = 125)
 
Women with placental blood infection (n = 145)
 
Variable SP group (n = 38) Placebo group (n = 87) P SP group (n = 47) Placebo group (n = 98) P 
Age, mean years (±SD)a 22 (6) 23 (6) .519 22 (6) 22 (6) .994 
Primigravidaeb 17 (45) 28 (32) .225 21 (45) 33 (34) .206 
HIV positiveb 15 (39) 26 (30) .304 19 (40) 26 (27) .124 
qPCR mean parasites/μL (±SD)a 18.7 (92.7) 90.7 (306.3) .041 9.4 (50.1) 225.8 (898.6) <.001 
Submicroscopic infectionb 16 (42) 49 (56) .174 26 (55) 55 (56) >.99 
MOI, mean value (±SD)a 2.6 (1.8) 3.0 (2.0) .333 2.5 (1.8) 3.1 (1.9) .119 
 Women with peripheral blood infection (n = 125)
 
Women with placental blood infection (n = 145)
 
Variable SP group (n = 38) Placebo group (n = 87) P SP group (n = 47) Placebo group (n = 98) P 
Age, mean years (±SD)a 22 (6) 23 (6) .519 22 (6) 22 (6) .994 
Primigravidaeb 17 (45) 28 (32) .225 21 (45) 33 (34) .206 
HIV positiveb 15 (39) 26 (30) .304 19 (40) 26 (27) .124 
qPCR mean parasites/μL (±SD)a 18.7 (92.7) 90.7 (306.3) .041 9.4 (50.1) 225.8 (898.6) <.001 
Submicroscopic infectionb 16 (42) 49 (56) .174 26 (55) 55 (56) >.99 
MOI, mean value (±SD)a 2.6 (1.8) 3.0 (2.0) .333 2.5 (1.8) 3.1 (1.9) .119 

NOTE. Data are no. (%) of women, unless otherwise indicated. HIV, human immunodeficiency virus; MOI, multiplicity of infection; qPCR, quantitative polymerase chain reaction; SD, standard deviation; SP, sulphadoxine-pyrimethamine.

a

Student's t test.

b

Fisher's exact test.

Effect of IPTp, Parity, Age, and HIV Status on the Prevalence of Q-Mutants at Delivery

In a preliminary survey of 60 samples collected in this study, all but 1 were fully wild-type at DHPS 581. As a result, only the dhfr 51, 59, 108 and dhps 437 and 540 genotypes were evaluated. The overall prevalence of q-mutant infections in the 270 peripheral and placental blood isolates was 12% (23 of 185 isolates) among pregnant women who received placebo and 24% (20 of 85 isolates) among those who received SP (P = .031). In the 125 peripheral blood isolates, the prevalence of mutant infections was similar between treatment groups (placebo group, 11 [13%] of 87; SP group, 6 [16%] of 38; P = .777), by parity (PG group, 7 [16%] of 45; MG group, 10 [13%] of 80; P = .786) and across age groups (P = .806), but it tended to be higher in HIV-positive women (9 [22%] of 41), compared with HIV-negative women (8 [10%] of 84; P = .092). On the other hand, the prevalence of q-mutant infections among the 145 placental blood isolates was higher in those from women who had received SP (14 [30%] of 47) than in those from placebo recipients (12 [12%] of 98; P = .019). It was also higher in isolates from HIV-positive women (13 [29%] of 45) than in isolates from HIV-negative women (13 [13%] of 100; P = .034). No difference in the prevalence of placental q-mutants was found between the PG group (10 [19%] of 54) and the MG group (16 [18%] and 91; P >.99) or across age groups (P = .927).

To take into account the interval between sample collection at delivery and the last SP dose, the analysis was stratified by early and late IPTp treatment (Figure 1). Placental q-mutants were significantly more prevalent in SP recipients (7 [50%] of 14) than in placebo recipients (6 [12%] of 52; P = .004) but only among women who received a late-IPTp intervention. In contrast, the prevalence of peripheral q-mutants was similar in SP (1 [7%] of 14) and placebo late recipients (4 [8%] of 51; P >.99). In both placental and peripheral infections, the prevalence of q-mutants was higher in HIV-positive women than in HIV-negative women who received late IPTp (Figure 1). No difference was found by parity (P >.99 for peripheral and placental blood infections) or across age groups (P = .848 for peripheral and P = .794 for placental blood infections). The multivariate analysis (Table 2) confirmed that treatment and HIV status were independently associated with higher risk of placental quintuple infections among women who received late IPTp intervention. No difference in the prevalence of q-mutants was found by intervention, HIV status, parity, or age among women who received early IPTp in the univariate (Figure 1) or in the multivariate (Table 2) analyses.

Table 2.

Multivariate Analysis Stratified by Early and Late Intermittent Preventive Treatment during Pregnancy (IPTp) showing the Associations between Prevalence of Quintuple Mutants at Delivery and IPTp, Parity, Age, and Human Immunodeficiency Virus (HIV) Status

   Periphery
 
Placenta
 
   OR 95% CI Pa OR 95%CI Pa 
≤ 2.5 months between last SP dose and delivery (late IPTp)
 IPTp Placebo     
  SP 0.85 0.08–9.31 .894 8.13 1.69–39.08 .009 
 Parity PG     
  MG 0.35 0.02–5.15 .443 1.28 0.19–8.52 .798 
 HIV status Negative      
  Positive NA NA NA 5.17 1.23–21.66 .025 
 Age, years ≤20     
  2125 4.18 0.26–61.32 0.566 0.46 0.13–1.70 .491 
  >25 4.59 0.15–144.65  0.67 0.15–2.97  
> 2.5 months between last SP dose and delivery (early IPTp)   
 IPTp Placebo     
  SP 1.26 0.33–4.80 0.737 2.00 0.57–7.01 .277 
 Parity PG     
  MG 0.80 0.11–6.02 0.831 1.55 0.23–10.70 .656 
 HIV status Negative     
  Positive 1.41 0.33–6.13 0.643 1.81 0.48–6.92 .383 
 Age, years ≤20     
  2125 0.3 0.04–2.31 0.391 1.30 0.20–8.26 .958 
  >25 0.95 0.10–9.12  1.11 0.10–12.47  
   Periphery
 
Placenta
 
   OR 95% CI Pa OR 95%CI Pa 
≤ 2.5 months between last SP dose and delivery (late IPTp)
 IPTp Placebo     
  SP 0.85 0.08–9.31 .894 8.13 1.69–39.08 .009 
 Parity PG     
  MG 0.35 0.02–5.15 .443 1.28 0.19–8.52 .798 
 HIV status Negative      
  Positive NA NA NA 5.17 1.23–21.66 .025 
 Age, years ≤20     
  2125 4.18 0.26–61.32 0.566 0.46 0.13–1.70 .491 
  >25 4.59 0.15–144.65  0.67 0.15–2.97  
> 2.5 months between last SP dose and delivery (early IPTp)   
 IPTp Placebo     
  SP 1.26 0.33–4.80 0.737 2.00 0.57–7.01 .277 
 Parity PG     
  MG 0.80 0.11–6.02 0.831 1.55 0.23–10.70 .656 
 HIV status Negative     
  Positive 1.41 0.33–6.13 0.643 1.81 0.48–6.92 .383 
 Age, years ≤20     
  2125 0.3 0.04–2.31 0.391 1.30 0.20–8.26 .958 
  >25 0.95 0.10–9.12  1.11 0.10–12.47  

NOTE. CI, confidence interval; MG, Multigravidae; NA: Not applicable due to lack of mutated infections among HIV-negative women; OR, odds ratio; PG, primigravidae; SP, sulphadoxine-pyrimethamine.

a

Logistic regression analysis.

b

n = 65 for peripheral; n = 66 for placental.

c

n = 60 for peripheral; n = 79 for placental.

Figure 1.

Prevalence of quintuple mutant Plasmodium falciparum infections by intermittent preventive treatment during pregnancy (IPTp) and human immunodeficiency virus (HIV) status among women receiving late (A) and early IPTp (B). P is the statistical significance of the difference between proportion among groups evaluated by Fisher's exact test. Plac, placebo.

Figure 1.

Prevalence of quintuple mutant Plasmodium falciparum infections by intermittent preventive treatment during pregnancy (IPTp) and human immunodeficiency virus (HIV) status among women receiving late (A) and early IPTp (B). P is the statistical significance of the difference between proportion among groups evaluated by Fisher's exact test. Plac, placebo.

The paired analysis (McNemar's test) of the 99 matched placental-peripheral blood infections from the same pregnant women at delivery showed that prevalence of q-mutants was higher in the placenta (7 [23%] of 30) than in the matched peripheral blood specimens (2 [7%] of 30) from women who received a late IPTp intervention (P = .025). However, no difference was found among women who had an early IPTp intervention (placenta blood specimens, 12 [17%] of 69; peripheral blood specimens, 11 [16%] of 69; P = .763).

Effect of Q-Mutants on Maternal-Fetal Outcomes

There was no statistically significant association between the presence of q-mutants and parasite densities, placental inflammation, MOI, newborn weight, and hematocrit level of the mother at delivery and postpartum, of the cord blood, or of the children at 1 month of age (Table 3). Similarly, no effect was found on the number of clinical malaria episodes in mothers during the postpartum period or in their children during the first year of life (Table 3).

Table 3.

Descriptive and Multivariate Analysis of the Association between Quintuple Mutants and Malaria-Related Morbidity in Mothers and their Infants

  Infecting strains with
 
 
  <5 Mutations 5 Mutations Pa 
Peripheral parasites 
 qPCR-parasite density (n = 125), geometric mean (±SD) 42.2 ± 166.4 344.8 ± 1297.2 .106b 
 Microscopical-parasite density (n = 60), geometric mean (±SD) 3560.5 ± 7775.8 4733.9 ± 9992.5 .761b 
 MOI at delivery (n = 125), mean value (±SD) 2.9 ± 2.0 3.1 ± 1.7 .689b 
 Newborn weigth (n = 123), mean kg (±SD) 2.89 ± .49 2.93 ± .47 .989b 
 Maternal hematocrit at delivery (n = 119), mean value (±SD) 31.9 ± 4.7 33.6 ± 4.2 .085b 
 Newborn hematocrit (n = 112), mean value (±SD) 44.7 ± 6.6 43.8 ± 6.4 .595b 
 Maternal hematocrit at postpartum (n = 96), mean value (±SD) 34.8 ± 3.9 33.9 ± 4.3 .896b 
 Children’s hematocrit at 1 month (n = 96), mean value (±SD) 37.0 ± 4.5 34.4 ± 11.0 .118b 
 No. of episodes during postpartum (n = 125), n/N (%) 7/108 (6) 1/17 (6) .684c 
 No. of episodes in children (n = 125), n/N (%) 55/108 (51) 5/17 (29) .502c 
Placental parasites 
 qPCR parasite density (n = 145), geometric mean value (±SD) 65.6 ± 294 206.4 ± 1143 .101b 
 Microscopic parasite density (n = 64), geometric mean value (±SD) 6737.3 ± 15008.6 8957.0 ± 18323.9 .639b 
 MOI at delivery (n = 145), mean value (±SD) 2.9 ± 2.0 2.7 ± 1.3 .126b 
 Placental inflammation (n = 145), n/N (%) 75/119 (63) 19/26 (73) .436d 
 Newborn weight (n = 144), mean kg (±SD) 2.94 ± .49 2.79 ± .56 .221b 
 Maternal hematocrit at delivery (n = 127), mean value (±SD) 32.3 ± 4.6 31.6 ± 4.2 .451b 
 Newborn hematocrit (n = 129), mean value (±SD) 44.9 ± 7.1 43.5 ± 7.2 .418b 
 Maternal hematocrit at postpartum (n = 92), mean value (±SD) 34.7 ± 3.9 33.8 ± 5.6 .365b 
 Children’s hematocrit at 1 month (n = 115), mean value (±SD) 36.9 ± 6.5 35.2 ± 9.8 .214b 
 No. of episodes during postpartum (n = 145), n/N (%) 7/119 (16) 0/26 (0) NA 
 No. of episodes in children (n = 145), n/N (%) 65/119 (55) 9/26 (35) .340c 
  Infecting strains with
 
 
  <5 Mutations 5 Mutations Pa 
Peripheral parasites 
 qPCR-parasite density (n = 125), geometric mean (±SD) 42.2 ± 166.4 344.8 ± 1297.2 .106b 
 Microscopical-parasite density (n = 60), geometric mean (±SD) 3560.5 ± 7775.8 4733.9 ± 9992.5 .761b 
 MOI at delivery (n = 125), mean value (±SD) 2.9 ± 2.0 3.1 ± 1.7 .689b 
 Newborn weigth (n = 123), mean kg (±SD) 2.89 ± .49 2.93 ± .47 .989b 
 Maternal hematocrit at delivery (n = 119), mean value (±SD) 31.9 ± 4.7 33.6 ± 4.2 .085b 
 Newborn hematocrit (n = 112), mean value (±SD) 44.7 ± 6.6 43.8 ± 6.4 .595b 
 Maternal hematocrit at postpartum (n = 96), mean value (±SD) 34.8 ± 3.9 33.9 ± 4.3 .896b 
 Children’s hematocrit at 1 month (n = 96), mean value (±SD) 37.0 ± 4.5 34.4 ± 11.0 .118b 
 No. of episodes during postpartum (n = 125), n/N (%) 7/108 (6) 1/17 (6) .684c 
 No. of episodes in children (n = 125), n/N (%) 55/108 (51) 5/17 (29) .502c 
Placental parasites 
 qPCR parasite density (n = 145), geometric mean value (±SD) 65.6 ± 294 206.4 ± 1143 .101b 
 Microscopic parasite density (n = 64), geometric mean value (±SD) 6737.3 ± 15008.6 8957.0 ± 18323.9 .639b 
 MOI at delivery (n = 145), mean value (±SD) 2.9 ± 2.0 2.7 ± 1.3 .126b 
 Placental inflammation (n = 145), n/N (%) 75/119 (63) 19/26 (73) .436d 
 Newborn weight (n = 144), mean kg (±SD) 2.94 ± .49 2.79 ± .56 .221b 
 Maternal hematocrit at delivery (n = 127), mean value (±SD) 32.3 ± 4.6 31.6 ± 4.2 .451b 
 Newborn hematocrit (n = 129), mean value (±SD) 44.9 ± 7.1 43.5 ± 7.2 .418b 
 Maternal hematocrit at postpartum (n = 92), mean value (±SD) 34.7 ± 3.9 33.8 ± 5.6 .365b 
 Children’s hematocrit at 1 month (n = 115), mean value (±SD) 36.9 ± 6.5 35.2 ± 9.8 .214b 
 No. of episodes during postpartum (n = 145), n/N (%) 7/119 (16) 0/26 (0) NA 
 No. of episodes in children (n = 145), n/N (%) 65/119 (55) 9/26 (35) .340c 

NOTE. IPTp, intermittent preventive treatment during pregnancy; MOI, multiplicity of infection; NA, not applicable; qPCR, quantitative polymerase chain reaction; SD, standard deviation.

a

Regression analysis adjusted by age, parity, IPTp, and period of last IPTp dose intake.

b

Linear regression.

c

Poisson regression.

d

Logistic regression.

DISCUSSION

Understanding the shifting patterns of SP resistance in its role for IPTp is vital to inform antimalarial policies in countries in which malaria is endemic. The results of this study show that, as long as residual SP was expected to have been cleared from the blood at delivery (if last IPTp dose was taken beyond 2.5 months before delivery) [9], the prevalence of placental and peripheral q-mutant parasites were similar in women who had received SP and those who had received placebo. However, IPTp was associated with an increase in q-mutant parasites in the placenta, but not in peripheral blood, when SP was expected to be present in blood (if last IPTp dose was taken within 2.5 months before delivery). Importantly, mutant infections were not associated with more-severe infections or higher malaria-related morbidity in mothers and their children.

This study shows that, although SP concentrations are expected to be uniform in the placenta and peripheral blood, the prevalence of highly drug-resistant parasites is higher in the placenta than in matched peripheral blood specimens. This observation cannot be explained by an increased selection of mutations in the placenta as a result of higher parasite densities [23], because a similar prevalence of mutated parasites in the peripheral and placental blood would be expected, given the overlapping distribution of genotypes in both compartments [24]. Our data, rather, support a model wherein q-mutants selected by SP can multiply in the placenta, because local immunity, likely altered to support the fetal allograft [25–27], clears parasites less effectively and therefore imposes less selective pressure on resistant parasites. In contrast, resistant parasites, with a deficient folate metabolism imposed by the mutations [28], may be killed by a more effective systemic immunity in the peripheral blood. This theory is consistent with our other finding, that q-mutants are also more prevalent in HIV-positive women, for whom adaptive immunity to P. falciparum is systemically impaired [5]. Because Mozambican policy for antenatal care for HIV-positive women at the time of the study included nevirapine but not the antifolate cotrimoxazole, confounding by cross-resistance is unlikely. We cannot discard, however, the possibility that folic acid supplementation, which is recommended to African pregnant women to prevent maternal anemia and which has been suggested to reduce clearance of P. falciparum by SP [29], might have influenced development of SP resistance in parasites from the groups that we compared. Our findings, together with the lower prevalence of q-mutants among pregnant women who received placebo as IPTp (16%), compared with that among children who also received placebo as IPTi (52%) [30] in the same area and period of time, support the notion that host immunity and fitness loss contribute importantly to the elimination of drug-resistant parasites [6, 31].

The current study shows that infections with q-mutant parasites are not associated with higher parasite densities or placental inflammation. This finding is in accordance with similar observations in the context of IPTi [30, 32] but contrasts with a recent report showing increased parasite densities and placental inflammation among Tanzanian women who received IPTp, an observation attributed to competitive facilitation [15]. The different findings could be explained if other genotypes, like mutations in codon dhps-581 [33], were responsible for higher levels of parasite resistance. However, in contrast to that study [15], this mutation was nearly absent from the parasite population infecting pregnant women from Manhiça area. Alternatively, lack of randomization and self-reported IPTp use in the first study [15] might have created endogeneity bias if women who chose IPTp were those with increased susceptibility to high-density malaria infections. Also, other mutations not assessed in our study might be associated with higher parasite densities in the placenta. Regardless of mutation status, SP was not found to increase parasitemias but, rather, was found to reduce qPCR-detected parasitemias and the MOI. Moreover, IPTp with SP has been shown to reduce the prevalence of peripheral and active placental infections [17]. Finally, this study showed that, as previously reported [12, 13], q-mutants were not associated with poor maternal-fetal outcomes or higher incidences of malaria episodes in women at postpartum or their infants, which is consistent with the lack of an effect of the mutations on parasite densities.

The results of this study suggest that the contribution of IPTp with SP to drug pressure at the population level is likely to be small. The IPTp-associated increase of q-mutant parasites in the placenta rapidly disappears after SP is expected to have been cleared from blood (2.5 months after drug intake), suggesting that these mutants are not competitive in the face of reinfection with other strains and that resistance will wane as active drug pressure decreases [28, 34]. A similar reduction in the prevalence of molecular markers of CQ resistance was observed in Malawi after CQ was abandoned as a first-line treatment [35]. Furthermore, the overall pressure exerted by SP given for IPTp is expected to be low, because pregnant women make up only 3% of the overall population of Manhiça (as in similar areas) at a given time. Finally, as SP alone is no longer a first-line antimalarial drug in areas of endemicity, treatment of pregnant women who show symptomatic malaria with artemisinin-based combination therapies should reduce the spread of mutant parasites.

It can be concluded that SP given for IPTp selects for dhfr/dhps mutant parasites in the placenta and in HIV-infected women, but only while residual plasma drug levels are present in blood. These results support the concept that impaired adaptive immune responses in the placenta [36] and in HIV-infected pregnant women [5] contribute to increased P. falciparum parasitemias [1, 37]. Importantly, mutant infections wane rapidly after SP is eliminated from blood and are not translated into more-severe infections or higher malaria-related morbidity in mothers and children. It is thus likely that IPTp with SP has only a subtle effect on SP resistance at the population level, especially in areas of endemicity, where competition from less-resistant parasites is intense and SP is not used as first-line therapy.

We thank the women participating in the study; the staff of the Manhiça District Hospital, clinical officers, field supervisors, and the data manager; and Cleofé Romagosa, Laura Puyol, Alfons Jiménez, Lázaro Mussacate, Nelito Ernesto José, Ana Rosa Manhiça, and Vania Simango, for their contribution to the collection and analysis of samples.

Financial support. Banco de Bilbao, Vizcaya, Argentaria Foundation (grant BBVA 02-0), Instituto de Salud Carlos III (grant PS09/01113, contract CP-04/00220 [to A.M.], and grant BF-03/00106 [to E.S.]), the Spanish Agency for International Cooperation, and the Ministerio de Ciencia e Innovación (RYC-2008-02631 to C.D.).

Potential conflicts of interest. All authors: no conflicts.

References

1.
Brabin
BJ
An analysis of malaria in pregnancy in Africa
Bull World Health Organ
 , 
1983
, vol. 
61
 
6
(pg. 
1005
-
1016
)
2.
White
NJ
McGready
RM
Nosten
FH
New medicines for tropical diseases in pregnancy: catch-22
PLoS Med
 , 
2008
, vol. 
5
 
6
pg. 
e133
 
3.
Ward
SA
Sevene
EJ
Hastings
IM
Nosten
F
McGready
R
Antimalarial drugs and pregnancy: safety, pharmacokinetics, and pharmacovigilance
Lancet Infect Dis
 , 
2007
, vol. 
7
 
2
(pg. 
136
-
144
)
4.
Yamada
M
Steketee
R
Abramowsky
C
, et al.  . 
Plasmodium falciparum associated placental pathology: a light and electron microscopic and immunohistologic study
Am J Trop Med Hyg
 , 
1989
, vol. 
41
 
2
(pg. 
161
-
168
)
5.
Mount
AM
Mwapasa
V
Elliott
SR
, et al.  . 
Impairment of humoral immunity to Plasmodium falciparum malaria in pregnancy by HIV infection
Lancet
 , 
2004
, vol. 
363
 
9424
(pg. 
1860
-
1867
)
6.
Rogerson
SJ
Wijesinghe
RS
Meshnick
SR
Host immunity as a determinant of treatment outcome in Plasmodium falciparum malaria
Lancet Infect Dis
 , 
2010
, vol. 
10
 
1
(pg. 
51
-
59
)
7.
Menendez
C
Bardaji
A
Sigauque
B
, et al.  . 
Malaria prevention with IPTp during pregnancy reduces neonatal mortality
PLoS One
 , 
2010
, vol. 
5
 
2
e9438
8.
World Health Organization (WHO)
A strategic framework for malaria prevention and control during pregnancy in the African region
 , 
2004
Brazzaville, Congo
WHO Regional Office for Africa
9.
Karunajeewa
HA
Salman
S
Mueller
I
, et al.  . 
Pharmacokinetic properties of sulfadoxine-pyrimethamine in pregnant women
Antimicrob Agents Chemother
 , 
2009
, vol. 
53
 
10
(pg. 
4368
-
4376
)
10.
Sibley
CH
Hyde
JE
Sims
PF
, et al.  . 
Pyrimethamine-sulfadoxine resistance in Plasmodium falciparum: what next?
Trends Parasitol
 , 
2001
, vol. 
17
 
12
(pg. 
582
-
588
)
11.
Kublin
JG
Dzinjalamala
FK
Kamwendo
DD
, et al.  . 
Molecular markers for failure of sulfadoxine-pyrimethamine and chlorproguanil-dapsone treatment of Plasmodium falciparum malaria
J Infect Dis
 , 
2002
, vol. 
185
 
3
(pg. 
380
-
388
)
12.
Mockenhaupt
FP
Bedu-Addo
G
Junge
C
Hommerich
L
Eggelte
TA
Bienzle
U
Markers of sulfadoxine-pyrimethamine-resistant Plasmodium falciparum in placenta and circulation of pregnant women
Antimicrob Agents Chemother
 , 
2007
, vol. 
51
 
1
(pg. 
332
-
334
)
13.
Mockenhaupt
FP
Bedu-Addo
G
Eggelte
TA
, et al.  . 
Rapid increase in the prevalence of sulfadoxine-pyrimethamine resistance among Plasmodium falciparum isolated from pregnant women in Ghana
J Infect Dis
 , 
2008
, vol. 
198
 
10
(pg. 
1545
-
1549
)
14.
Bouyou-Akotet
MK
Mawili-Mboumba
DP
Tchantchou Tde
D
Kombila
M
High prevalence of sulfadoxine/pyrimethamine-resistant alleles of Plasmodium falciparum isolates in pregnant women at the time of introduction of intermittent preventive treatment with sulfadoxine/pyrimethamine in Gabon
J Antimicrob Chemother
 , 
2010
, vol. 
65
 
3
(pg. 
438
-
441
)
15.
Harrington
WE
Mutabingwa
TK
Muehlenbachs
A
, et al.  . 
Competitive facilitation of drug-resistant Plasmodium falciparum malaria parasites in pregnant women who receive preventive treatment
Proc Natl Acad Sci U S A
 , 
2009
, vol. 
106
 
22
(pg. 
9027
-
9032
)
16.
Alonso
P
Saute
F
Aponte
JJ
, et al.  . 
Manhica demographic surveillance System, Mozambique
Population, Health and Survival at INDEPTH Sites
 , 
2001
, vol. 
1
 (pg. 
189
-
195
)
17.
Menendez
C
Bardaji
A
Sigauque
B
, et al.  . 
A randomized placebo-controlled trial of intermittent preventive treatment in pregnant women in the context of insecticide treated nets delivered through the antenatal clinic
PLoS One
 , 
2008
, vol. 
3
 
4
pg. 
e1934
 
18.
Alonso
PL
Smith
T
Schellenberg
JR
, et al.  . 
Randomised trial of efficacy of SPf66 vaccine against Plasmodium falciparum malaria in children in southern Tanzania
Lancet
 , 
1994
, vol. 
344
 
8931
(pg. 
1175
-
1181
)
19.
Ismail
MR
Ordi
J
Menendez
C
, et al.  . 
Placental pathology in malaria: a histological, immunohistochemical, and quantitative study
Hum Pathol
 , 
2000
, vol. 
31
 
1
(pg. 
85
-
93
)
20.
Mayor
A
Serra-Casas
E
Bardaji
A
, et al.  . 
Sub-microscopic infections and long-term recrudescence of Plasmodium falciparum in Mozambican pregnant women
Malar J
 , 
2009
, vol. 
8
 pg. 
9
 
21.
Plowe
CV
Cortese
JF
Djimde
A
, et al.  . 
Mutations in Plasmodium falciparum dihydrofolate reductase and dihydropteroate synthase and epidemiologic patterns of pyrimethamine-sulfadoxine use and resistance
J Infect Dis
 , 
1997
, vol. 
176
 
6
(pg. 
1590
-
1596
)
22.
Snounou
G
Zhu
X
Siripoon
N
, et al.  . 
Biased distribution of msp1 and msp2 allelic variants in Plasmodium falciparum populations in Thailand
Trans R Soc Trop Med Hyg
 , 
1999
, vol. 
93
 
4
(pg. 
369
-
374
)
23.
White
NJ
Pongtavornpinyo
W
The de novo selection of drug-resistant malaria parasites
Proc Biol Sci
 , 
2003
, vol. 
270
 
1514
(pg. 
545
-
554
)
24.
Jafari-Guemouri
S
Ndam
NT
Bertin
G
, et al.  . 
Demonstration of a high level of parasite population homology by quantification of Plasmodium falciparum alleles in matched peripheral, placental, and umbilical cord blood samples
J Clin Microbiol
 , 
2005
, vol. 
43
 
6
(pg. 
2980
-
2983
)
25.
Diouf
I
Fievet
N
Doucoure
S
, et al.  . 
IL-12 producing monocytes and IFN-gamma and TNF-alpha producing T-lymphocytes are increased in placentas infected by Plasmodium falciparum
J Reprod Immunol
 , 
2007
, vol. 
74
 
1–2
(pg. 
152
-
162
)
26.
Diouf
I
Fievet
N
Doucoure
S
, et al.  . 
Monocyte activation and T cell inhibition in Plasmodium falciparum-infected placenta
J Infect Dis
 , 
2004
, vol. 
189
 
12
(pg. 
2235
-
2242
)
27.
Riley
EM
Schneider
G
Sambou
I
Greenwood
BM
Suppression of cell-mediated immune responses to malaria antigens in pregnant Gambian women
Am J Trop Med Hyg
 , 
1989
, vol. 
40
 
2
(pg. 
141
-
144
)
28.
Hastings
IM
Donnelly
MJ
The impact of antimalarial drug resistance mutations on parasite fitness, and its implications for the evolution of resistance
Drug Resist Updat
 , 
2005
, vol. 
8
 
1–2
(pg. 
43
-
50
)
29.
Carter
JY
Loolpapit
MP
Lema
OE
Tome
JL
Nagelkerke
NJ
Watkins
WM
Reduction of the efficacy of antifolate antimalarial therapy by folic acid supplementation
Am J Trop Med Hyg
 , 
2005
, vol. 
73
 
1
(pg. 
166
-
170
)
30.
Mayor
A
Serra-Casas
E
Sanz
S
, et al.  . 
Molecular markers of resistance to sulfadoxine-pyrimethamine during intermittent preventive treatment for malaria in Mozambican infants
J Infect Dis
 , 
2008
, vol. 
197
 
12
(pg. 
1737
-
1742
)
31.
Djimde
AA
Doumbo
OK
Traore
O
, et al.  . 
Clearance of drug-resistant parasites as a model for protective immunity in Plasmodium falciparum malaria
Am J Trop Med Hyg
 , 
2003
, vol. 
69
 
5
(pg. 
558
-
563
)
32.
Marks
F
von Kalckreuth
V
Kobbe
R
, et al.  . 
Parasitological rebound effect and emergence of pyrimethamine resistance in Plasmodium falciparum after single-dose sulfadoxine-pyrimethamine
J Infect Dis
 , 
2005
, vol. 
192
 
11
(pg. 
1962
-
1965
)
33.
Gesase
S
Gosling
RD
Hashim
R
, et al.  . 
High resistance of Plasmodium falciparum to sulphadoxine/pyrimethamine in northern Tanzania and the emergence of dhps resistance mutation at Codon 581
PLoS One
 , 
2009
, vol. 
4
 
2
pg. 
e4569
 
34.
Chawira
AN
Warhurst
DC
Peters
W
Qinghaosu resistance in rodent malaria
Trans R Soc Trop Med Hyg
 , 
1986
, vol. 
80
 
3
(pg. 
477
-
480
)
35.
Nkhoma
S
Molyneux
M
Ward
S
Molecular surveillance for drug-resistant Plasmodium falciparum malaria in Malawi
Acta Trop
 , 
2007
, vol. 
102
 
2
(pg. 
138
-
142
)
36.
Wegmann
TG
Lin
H
Guilbert
L
Mosmann
TR
Bidirectional cytokine interactions in the maternal-fetal relationship: is successful pregnancy a TH2 phenomenon?
Immunol Today
 , 
1993
, vol. 
14
 
7
(pg. 
353
-
356
)
37.
Steketee
RW
Wirima
JJ
Bloland
PB
, et al.  . 
Impairment of a pregnant woman's acquired ability to limit Plasmodium falciparum by infection with human immunodeficiency virus type-1
Am J Trop Med Hyg
 , 
1996
, vol. 
55
 
suppl 1
(pg. 
42
-
49
)

Comments

0 Comments