Malaria Branch, Division of Parasitic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; 2

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1 MAJOR ARTICLE Randomized Trial of 2-Dose versus Monthly Sulfadoxine-Pyrimethamine Intermittent Preventive Treatment for Malaria in HIV-Positive and HIV-Negative Pregnant Women in Malawi Scott J. Filler, 1,a Peter Kazembe, 2 Michael Thigpen, 1,a Alan Macheso, 2 Monica E. Parise, 1 Robert D. Newman, 1 Richard W. Steketee, 1,a and Mary Hamel 1 1 Malaria Branch, Division of Parasitic Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; 2 Malawi Ministry of Health and Population, Lilongwe, Malawi (See the editorial commentary by Meshnick et al., on pages ) Background. Intermittent preventive treatment during pregnancy (IPTp) with sulfadoxine-pyrimethamine(sp) decreases placental malaria parasitemia and associated maternal anemia, premature delivery, and low birth weight. However, the optimal regimen in the setting of a high prevalence of human immunodeficiency virus (HIV) infection remains unclear. Methods. In Malawi, where the efficacy of SP for the treatment of malaria in children is decreasing, we conducted a randomized, nonblinded study to compare the efficacy of monthly SP IPTp with a 2-dose regimen for the prevention of placental parasitemia in HIV-positive and -negative primigravid and secundigravid women. Results. Of HIV-positive women, 7.8% who received monthly SP had placental malaria, compared with 21.5% of those who received 2-dose SP (relative risk [RR], 0.36 [95% confidence interval {CI}, ]). Of HIVnegative women, 2.3% who received monthly SP and 6.3% who received 2-dose SP had placental malaria (RR, 0.37 [95% CI, ]). Less than 1% of women reported adverse drug reactions, with no increase in HIVpositive women or those who received monthly SP. Conclusions. In HIV-positive pregnant women, monthly SP IPTp is more efficacious than a 2-dose regimen in preventing placental malaria. The study also demonstrates the continued efficacy of SP for the prevention of placental malaria, even in the face of its decreasing efficacy for the treatment of malaria in children. In areas with intense transmission of falciparum malaria and a high prevalence of HIV infection, monthly SP IPTp should be adopted. Malaria has a devastating impact in sub-saharan Africa, where an estimated million cases and 1 million deaths occur annually [1]. Each year, 25 million African Received 30 November 2005; accepted 1 March 2006; electronically published 20 June Potential conflicts of interest: none reported. Financial support: World Health Organization; Malaria Branch, Division of Parasitic Diseases, US Centers for Disease Control and Prevention. Clinical trial registry s URL: Clinical trial registration number: NCT a Present affiliations: Care and Treatment Branch, Global AIDS Program (S.F.), and Surveillance and Epidemiology Branch, Division of HIV/AIDS Prevention (M.T.), Centers for Disease Control and Prevention, Atlanta, Georgia; Malaria Control and Evaluation Program in Africa, Program for Appropriate Technology in Health, Seattle, Washington (R.W.S.). Reprints or correspondence: Dr. Scott Filler, Centers for Disease Control and Prevention, 1600 Clifton Rd., Mailstop E-04, Atlanta, GA, (SFiller@cdc.gov). The Journal of Infectious Diseases 2006; 194: by the Infectious Diseases Society of America. All rights reserved /2006/ $15.00 women become pregnant in areas where malaria is endemic [2]. In areas of stable malaria transmission, pregnant women with Plasmodium falciparum infection are often asymptomatic, but the parasite can sequester in the placenta. These infections are associated with maternal anemia, premature delivery, and low birth weight (LBW;!2500 g) [2 7]. Primigravid and secundigravid women have the highest prevalence of placental parasitemia and malaria-associated LBW [8 12]. LBW is the greatest risk factor for neonatal mortality and is a major contributor to infant mortality [13]. Malaria is one of the few contributors to LBW that is amenable to a specific intervention. The World Health Organization recommends that all women living in areas of sub-saharan Africa with stable malaria transmission receive intermittent preventive treatment during pregnancy (IPTp) with an effective 286 JID 2006:194 (1 August) Filler et al.

2 antimalarial drug during routine antenatal care (ANC) [2]. In Malawi, Schultz et al. [14] showed that a 2-dose IPTp regimen of sulfadoxine-pyrimethamine (SP) administered during the second and third trimesters of pregnancy reduced placental malaria at delivery. However, HIV infection has become increasingly prevalent among women of reproductive age in sub- Saharan Africa [15 17]. HIV infection diminishes a pregnant woman s capacity to control P. falciparum infection, resulting in a suboptimal response to 2-dose SP IPTp [18]. A study in Kenya demonstrated that more frequent SP dosing may provide further reductions in placental malaria, especially in HIV-infected women [4]. The previous studies in Malawi and Kenya that demonstrated the efficacy of SP IPTp were conducted when SP was highly efficacious for the treatment of uncomplicated malaria [4, 14]. In Malawi, as in many other East African countries, the efficacy of SP is decreasing, with a 36% risk of parasitological treatment failure by day 14 in children [19]. The efficacy of SP for IPTp under conditions of waning efficacy of SP for treatment is unknown. At the time of the present study, the Malawi Ministry of Health and Population s guidelines recommended 2-dose SP IPTp, administered during the second and third trimesters. Our primary study objective was to determine the efficacy of monthly SP, compared with that of the 2-dose regimen, in the prevention of placental malaria in HIV-positive women. HIVnegative women were included to determine the efficacy of both 2-dose and monthly SP IPTp and to decrease any stigma associated with being in the study. A secondary objective was to demonstrate the safety of monthly SP IPTp. SUBJECTS AND METHODS Institutional review. The institutional review boards of the Malawi Ministry of Health and Population and the US Centers for Disease Control and Prevention (CDC) approved the study protocol. Study site. We conducted the study from October 2002 through March 2005 at the Machinga District Hospital, Liwonde, Malawi, an area of intense P. falciparum transmission. SP is used for IPTp and as first-line treatment for uncomplicated malaria. Enrollment. We enrolled clinic patients seeking ANC in Machinga District Hospital. Women in their first and second pregnancies who were between 16 and 28 weeks of gestation were invited to participate. Study nurse midwives were trained HIV counselors fluent in the local languages. After the explanation of study procedures and receipt of signed informed consent, women underwent a physical examination and completed a questionnaire on demographics, socioeconomic status, malaria symptoms, antimalarial drug use, insecticide-treated bed net (ITN) use, and contact information. Women reporting a prior adverse drug reaction (ADR) to sulfacontaining medications or quinine were excluded. A venous blood sample was drawn for HIV testing, hemoglobin measurement, syphilis screening, and a malaria thick blood smear. Study nurse midwives provided pre- and posttest HIV counseling. CD4 + cell count testing and combination antiretroviral therapy were not routinely available in Malawi during the time of the trial. We estimated the seroprevalence of HIV to be 20% [20]; so that we might have approximately equal numbers of HIV-positive and -negative women in the study, 1 of every 4 HIV-negative clients was enrolled sequentially. Women were given 200 mg of ferrous sulfate and 0.5 mg of folic acid for daily self-administration. Intervention. Enrolled women were randomized, by permuted blocks of random length, to 1 of 2 IPTp regimens, by HIV status: (1) 2-dose SP, with directly observed treatment doses (1500 mg of sulfadoxine and 75 mg of pyrimethamine) at enrollment and at the first appointment after 28 weeks, in accordance with Ministry of Health and Population guidelines; and (2) monthly SP, with directly observed treatment doses at enrollment and then monthly until delivery. Blinding. Trained laboratory workers who assessed the primary outcome of placental malaria were blinded to the women s HIV status and treatment arm. Neither study participants nor clinicians were blinded to group assignment. Sample size. We estimated a target sample size of 350 HIVpositive women on the basis of a 95% confidence level (CI) and 90% power to detect a 15% difference in the prevalence of placental malaria between the 2 regimens. However, enrollment was slower than projected because of a lower than anticipated prevalence of HIV infection, and funds were limited. Therefore, in October 2004, we conducted an interim analysis to assess the primary outcome measure of placental parasitemia. The interim analysis assumed that 2 sequential tests would be made using the a-time spending function [21]. The nominal a-value for the first (interim) evaluation was 0.030, and the nominal a-value for the second (final) analysis was At the interim analysis, we found a significant difference in the prevalence of placental parasitemia between the 2 treatment regimens in HIV-positive women and felt that the study should be stopped. Therefore, we discontinued enrollment before reaching the target sample size. All women in the 2-dose arm at the time of the interim analysis received monthly SP for the remainder of their pregnancies. Follow-up. All women were seen monthly by study nurse midwives until delivery. At each monthly follow-up visit, women were asked about ADRs to SP, malaria symptoms, and routine obstetric issues. If a participant complained of malarial symptoms, a thick blood smear was examined. Women who failed to attend a scheduled monthly appointment were traced to their homes and, if they were willing to continue in the study, were brought to the clinic for a follow-up visit. He- Prevention of Malaria during Pregnancy JID 2006:194 (1 August) 287

3 Figure 1. Study population. CTX, cotrimoxazole; IPTp, intermittent preventive treatment during pregnancy; SP, sulfadoxine-pyrimethamine. moglobin was measured at the first visit after 28 weeks of gestation. Women were encouraged to give birth in the hospital, and the study team assisted with transportation when needed. Single-dose nevirapine was given to all HIV-positive women at 32 weeks of gestation to self-administer once they entered active labor. Women with symptomatic uncomplicated malaria received SP for treatment, except for those who presented!4 weeks after the last dose of SP and those randomized to the 2-dose IPTp arm who presented either too early for the scheduled second dose of SP or after their second dose. These women received quinine. Women were instructed to present to the study team for evaluation of any intercurrent illnesses. Women with illness or severe anemia detected at any time were seen by the study s clinical officer and treated in accordance with Ministry of Health and Population guidelines. Delivery. Thick blood smears were made from maternal capillary blood (via finger stick) and from the placenta and umbilical cord after delivery. Newborns were weighed on a 288 JID 2006:194 (1 August) Filler et al.

4 Table 1. Baseline characteristics of randomized women, by HIV status and treatment arm. Sulfadoxine-pyrimethamine regimen Characteristic HIV-positive women 2-dose (n p 131) Monthly (n p 135) HIV-negative women 2-dose (n p 216) Monthly (n p 216) Age, mean SD, years Primigravida Length of gestation, mean SD, weeks Ethnicity Yao Lomwe Chewa Other Completed primary school Married Slept under an ITN the previous night Height!150 cm Weight!50 kg Peripheral parasitemia Hb level, mean SD, g/dl Severe anemia (Hb!7 g/dl) NOTE. bed net. Data are percentage of women, unless otherwise indicated. Hb, hemoglobin; ITN, insecticide-treated digital scale, accurate to the nearest gram, within 24 h of birth. Gestational age was determined by physical and neurological examination, using a Ballard assessment tool [22]. Single-dose nevirapine was given to all HIV-positive women in active labor (if they had not appropriately self-administered a dose) and to newborns (with dose based on weight) of HIV-positive mothers within 72 h of birth. HIV-infected women who gave birth outside of the hospital were instructed to bring their infants to the study clinic as early as possible, so that single-dose nevirapine could be administered to the infants. Infant follow-up. General health status, including an examination for scleral icterus, was assessed in all newborns during a home or clinic visit between 3 and 7 days of life and again at age 1 month. Any infant showing signs of illness was referred to the hospital, and the study team assisted with transportation when necessary. An attempt was made to determine the cause of any neonatal deaths by questioning the mother about premortem symptoms. Laboratory investigations. Thick blood smears were stained with Giemsa and examined for P. falciparum. Malaria parasites and leukocytes were counted in the same fields until 300 leukocytes or 500 parasites were counted. Placental blood smears were prepared by making an incision of the maternal side, allowing the blood to pool, and then collecting the blood with a bulb pipette. A HemoCue detection system (HemoCue) was used to measure hemoglobin. HIV testing was performed using rapid tests in parallel (Uni-Gold; Trinity Biotech and Determine; Abbott Laboratories). All discordant results were further evaluated using a third rapid test (Hema Strip; Saliva Diagnostic Systems) and a confirmatory EIA. Definitions. Parasitemia (in peripheral, placental, or cord blood) was defined as the presence of asexual-stage parasites in thick smears. Newborns weighing!2500 g were considered to be of LBW. Neonates assessed as being at!37 weeks of gestation at birth (using the Ballard score) were classified as premature. Women with hemoglobin levels of!11 and!7 g/dl were considered to have anemia and severe anemia, respectively. Severe cutaneous ADRs were defined as erythema multiforme, Stevens- Johnson syndrome, or toxic epidermal necrolysis. Statistical analysis. Data were double-entered using Epi Info (version 6; CDC). Data validation, cleaning, and statistical analysis were performed using SAS software (version 6; SAS Institute). In the main analysis, women were excluded who started out in the 2-dose arm and then were given monthly SP after the interim analysis. In addition, a second, intention-totreat analysis was performed in which data were again analyzed according to original arm assignments but included women assigned to the 2-dose arm who were changed to monthly dosing because of the interim analysis. Third-trimester hemoglobin data were included for all women who had a measurement taken, even if their placentas were not available for analysis. Parasitemia results at the time of delivery included data from women whose placentas were analyzed. Results from women who gave birth to live singleton newborns were included in the Prevention of Malaria during Pregnancy JID 2006:194 (1 August) 289

5 Figure 2. Actual no. of doses of sulfadoxine-pyrimethamine delivered to women receiving monthly intermittent preventive treatment during pregnancy. analyses of birth weight, timing, and fetal death. Differences between means were compared using 1-way analysis of variance or the Kruskal-Wallis test. Differences between proportions were compared using the x 2 or Fisher s exact test. RESULTS Study population. Overall, 1892 women consented to participate in the trial and underwent HIV testing (figure 1). A total of 266 were identified as being HIV positive and randomized: 131 to receive 2 doses of SP and 135 to receive monthly SP. In all, 432 HIV-negative clients were enrolled and randomized: 216 to receive 2 doses of SP and 216 to receive monthly SP. The distribution of baseline characteristics was similar by treatment arm for both HIV-positive and -negative women (table 1). After the interim analysis revealed a significant difference in the prevalence of placental parasitemia between treatment arms for HIV-positive clients, 13 HIV-positive women in the 2-dose arm and 44 HIV-negative women in the 2-dose arm were crossed over to receive monthly SP. These 57 women were not included in the main analysis of delivery outcomes. A total of 143 (22%) of the women did not have a live birth in the hospital; 85 women (13%) gave birth at home, 31 (5%) moved from the study area, and 9 (1%) had spouses who refused to allow the participant to continue. The remaining 18 (3%) did not the complete the study for other reasons. Women who completed the study were not different from those who did not with respect to baseline characteristics. Of HIV-positive women in the monthly SP arm, 80% received 13 doses (median, 5 doses) (figure 2). Of HIV-negative women in the monthly SP arm, 85% received 13 doses (median, 5 doses). Of HIV-positive women, 56% in the 2-dose and 39% in the monthly arm reported sleeping under an ITN the previous night at any follow-up visit. Of HIV-negative women, 49% in the 2-dose and 51% in the monthly arm reported ever sleeping under an ITN the previous night at any follow-up visit. Efficacy of regimens. At delivery, HIV-positive women in the monthly SP arm were less likely to have placental malaria than were HIV-positive women in the 2-dose SP arm (relative risk [RR], 0.36 [95% CI, ]) (figure 3). HIV-negative women in the monthly SP arm were less likely to have placental malaria than were HIV-negative women in the 2-dose SP arm, but the difference did not reach statistical significance (RR, 0.37 [95% CI, ]). Results were similar for the additional intention-to-treat analysis. In this analysis, HIV-positive women in the monthly SP arm were less likely to have placental malaria than were HIV-positive women in the 2-dose SP arm (RR, 0.38 [95% CI, ]), and HIV-negative women in the monthly SP arm were less likely to have placental malaria than were HIV-negative women in the 2-dose SP arm, but the difference did not reach statistical significance (RR, 0.47 [95% CI, ]). Of HIV-positive women, 12% in the monthly and 35% in the 2-dose arm had at least 1 episode of symptomatic uncomplicated malaria (RR, 0.34 [95% CI, ]). Of HIV-negative women, 13% in the monthly and 25% in the 2-dose arm had at least 1 episode of symptomatic uncomplicated malaria (RR, 0.50 [95% CI, ]). ADRs: mothers. Overall,!1% of women reported ADRs (only rash, nausea, vomiting, and fever were reported), and in no case was SP withheld because of these concerns. There were no significant differences in the proportion of women reporting ADRs in the 2-dose and monthly SP arms when stratified by HIV status. No severe cutaneous ADRs were observed in 1734 treatment episodes among 641 women. ADRs: neonates. Neonatal jaundice was observed in 0.4% of the newborns examined between 3 and 7 days of life. Twenty deaths occurred in 486 newborns monitored through the neonatal period, with no increased frequency of death in either study arm (table 2). Of these 20 neonatal deaths, 1 infant born to an HIV-positive mother in the 2-dose SP arm had neonatal Figure 3. Placental parasitemia, by HIV status and treatment arm 290 JID 2006:194 (1 August) Filler et al.

6 Table 2. Outcomes, by HIV status and treatment arm. Sulfadoxine-pyrimethamine regimen Characteristic Third-trimester Hb a HIV-positive women HIV-negative women 2-dose Monthly P 2-dose Monthly P Mean SD, g/dl Anemia Severe anemia b b Parasitemia at delivery c Maternal peripheral blood ! Placental Cord blood b Birth weight and timing d BW, mean SD, kg LBW Premature delivery (!37 weeks) b b Fetal death e Spontaneous abortion b b Stillbirth b b Neonatal death within 30 days of birth d b b NOTE. Data are percentage of women, unless otherwise indicated. BW, birth weight; Hb, hemoglobin; LBW, low birth weight (!2.5 kg). a Includes data from women with known third-trimester Hb levels. Anemia, Hb level!11 g/dl; severe anemia, Hb level!7 g/dl. b Fisher s 2-tailed exact test. c Includes data from women whose placentas were analyzed. d Includes data from women who gave birth to live singleton infants. e Includes data from women with known birth outcomes. jaundice. The cause of death for this neonate was attributed to prematurity (30 weeks of gestation) and a LBW of!1 kg. DISCUSSION In the present randomized trial, which compared 2 regimens for the prevention of malaria during pregnancy, we demonstrated that, among HIV-positive primigravid and secundigravid women, IPTp with monthly SP was more efficacious than 2-dose SP IPTp in preventing placental malaria. The extremely low prevalence of placental parasitemia seen in HIVnegative women in the monthly SP arm and the low rate of ADRs indicate that monthly SP may be the best regimen for HIV-negative women as well. Such an approach would obviate the need for a more complicated program that provides differential regimens based on HIV status. Our results demonstrate the continued efficacy of SP IPTp for the prevention of malaria in semi-immune pregnant women, even in the face of the decreasing efficacy of SP for the treatment of malaria in young children. In Malawi, 2-dose IPTp with SP has been the national policy since During this period, there have been significant trends toward diminishing clinical and parasitological efficacy of SP for the treatment of children with uncomplicated malaria [23]. Given its low cost, wide availability, easy deliverability, safety, and now-demonstrated continued efficacy, SP should continue to be provided for IPTp. Many countries in sub-saharan Africa are changing first-line therapy for uncomplicated malaria away from chloroquine and SP toward combination therapy, particularly with artemisinin-based combination therapies (ACTs). However, until more data are available on the safety of ACTs and other drugs during pregnancy, SP should still be used for IPTp. The results of the present trial are consistent with findings from Kenya indicating that monthly SP IPTp is efficacious in HIV-positive pregnant women for the prevention of placental parasitemia [4]. Parise et al. found that the monthly regimen was superior to a case-management approach, but their study did not have the power to discern the difference between 2- dose SP IPTp and a monthly regimen. Our study, which included a larger sample of HIV-positive pregnant women, was able to further clarify the benefit of monthly SP IPTp. There were no significant differences in premature delivery, spontaneous abortion, stillbirth, or ADRs between the 2 treatment arms by HIV status, and there were no reported cases of kernicterus. The paucity of ADRs associated with SP and the lack of association with adverse maternal or fetal outcomes are also consistent with the findings of other published trials [4, 14, 24]. The mean singleton birth weight was higher and the incidence of LBW was lower for both HIV-positive and -negative women who received monthly SP, although these differences Prevention of Malaria during Pregnancy JID 2006:194 (1 August) 291

7 did not reach statistical significance. Large sample sizes are required to sufficiently examine the effect of a single factor on birth weight; therefore, because of the demonstrated association between placental malaria and LBW [12], our study was designed to use the prevalence of placental malaria as a surrogate measurement of the risk of LBW. The prevalence of severe anemia during the third trimester was lower in the monthly treatment arms, but this difference did not reach statistical significance. The ability to detect this difference also would have been difficult, given the low prevalence of severe anemia in the study population. Nonetheless, the direction of these associations further supports monthly dosing. Monthly SP dosing may be easier to implement programmatically than 2-dose IPTp. Despite 7 years of implementation of the national policy in Malawi, coverage of 2-dose SP IPTp was only 29.3% in 2000 [25]. A survey of health-care workers demonstrated that one of several reasons why SP IPTp was not provided according to national guidelines was confusion as to when SP should be provided and when it should be withheld [26]. A recent district-level survey showed that ANC attendance was 199% among primigravid and secundigravid women, with an average of 4 ANC visits per pregnant woman (national policy recommends monthly ANC visits) [27]. Thus, there have been extensive missed opportunities in which pregnant women have visited health-care facilities but have not received IPTp. A pilot project was initiated in the Blantyre district of Malawi in 2000 and 2001 to reduce confusion about when to provide SP IPTp. Health-care workers were instructed to give SP at every ANC visit after quickening, unless SP had been provided during the preceding 4 weeks. A follow-up survey demonstrated an increase in SP IPTp coverage to 79% [26]. Thus, providing SP at every ANC visit resulted in a rapid increase in SP IPTp coverage and may be a more successful intervention programmatically than 2- dose SP. The present study had limitations that need to be considered when interpreting the results. Participants and study staff were not blinded to study arm. Because the primary outcome was objective and examined by blinded laboratory workers, the added cost and complexity of administering placebos outweighed the concern of this potential bias. The lack of blinding may have affected ITN use. Among HIV-positive women, more women in the 2-dose arm reported ever sleeping under an ITN during follow-up. This may have represented differential care if patients and caretakers believed these patients to be at the highest risk for placental malaria among the 4 groups or this simply may have been a random occurrence. Regardless of etiology, for HIVpositive women, this difference would have biased the association toward the null hypothesis and, thus, further strengthens the findings of improved outcomes for monthly dosing. Despite extensive effort, another limitation of the trial was loss to follow-up. Deliveries at home (the main reason why data were not available) eliminated the opportunity for us to investigate the placenta. The extent to which the prevalence of placental malaria differed by loss to follow-up cannot be examined. However, because there were similar rates of loss to follow-up among the treatment groups by HIV status and because these women had similar baseline characteristics, compared with all who were enrolled, this is unlikely to have altered study results. Finally, because women in the monthly SP IPTp arm received doses late during pregnancy, the reduction in placental malaria may simply reflect clearance of the placenta close to delivery rather than cumulative protection during the course of gestation. However, if placental malaria is cleared with more frequency during late pregnancy with the monthly regimen, it is logical to conclude that monthly dosing clears the placenta during other vulnerable periods not covered by the 2-dose regimen. This is supported by the fact that women in the monthly treatment arm had significantly fewer episodes of symptomatic malaria than did those in the 2-dose arm. In conclusion, we have shown that SP IPTp continues to be efficacious even in an area of waning efficacy of SP for treatment and that monthly SP IPTp is more efficacious than 2-dose SP IPTp in HIV-positive women, and possibly in HIV-negative women, in decreasing the prevalence of placental malaria. Given the superior efficacy of monthly SP, improved coverage achieved by providing SP at all ANC visits rather than at specified visits, and the low frequency of ADRs with monthly administration of SP, a change from 2-dose IPTp to SP IPTp provided at every ANC visit should be promoted in this and similar settings. Acknowledgments We thank the medical and nursing staff of Machinga District Hospital, Liwonde, Malawi; the dedicated team of the Blantyre Integrated Malaria Initiative, Blantyre, Malawi (notably, Carl Campbell, Nyson Chinadzi, and Rachel Bronzan); and, most of all, the women who participated in the study. References 1. Hay SI, Guerra CA, Tatem AJ, Atkinson PM, Snow RW. Urbanization, malaria transmission and disease burden in Africa. Nat Rev Microbiol 2005; 3: World Health Organization (WHO). A strategic framework for malaria prevention and control during pregnancy in the African region: report AFR/MAL/04/01. 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