A randomized controlled trial of folate supplementation when treating malaria in pregnancy with sulfadoxine-pyrimethamine.

Abstract

Sulfadoxine-pyrimethamine (SP) is an antimalarial drug that acts on the folate metabolism of the malaria parasite. We investigated whether folate (FA) supplementation in a high or a low dose affects the efficacy of SP for the treatment of uncomplicated malaria in pregnant women.
This was a randomized, placebo-controlled, double-blind trial.
The trial was carried out at three hospitals in western Kenya.
The participants were 488 pregnant women presenting at their first antenatal visit with uncomplicated malaria parasitaemia (density of >or= 500 parasites/microl), a haemoglobin level higher than 7 g/dl, a gestational age between 17 and 34 weeks, and no history of antimalarial or FA use, or sulfa allergy. A total of 415 women completed the study.
All participants received SP and iron supplementation. They were randomized to the following arms: FA 5 mg, FA 0.4 mg, or FA placebo. After 14 days, all participants continued with FA 5 mg daily as per national guidelines. Participants were followed at days 2, 3, 7, 14, 21, and 28 or until treatment failure.
The outcomes were SP failure rate and change in haemoglobin at day 14.
The proportion of treatment failure at day 14 was 13.9% (19/137) in the placebo group, 14.5% (20/138) in the FA 0.4 mg arm (adjusted hazard ratio [AHR], 1.07; 98.7% confidence interval [CI], 0.48 to 2.37; p = 0.8), and 27.1% (38/140) in the FA 5 mg arm (AHR, 2.19; 98.7% CI, 1.09 to 4.40; p = 0.005). The haemoglobin levels at day 14 were not different relative to placebo (mean difference for FA 5 mg, 0.17 g/dl; 98.7% CI, -0.19 to 0.52; and for FA 0.4 mg, 0.14 g/dl; 98.7% CI, -0.21 to 0.49).
Concomitant use of 5 mg FA supplementation compromises the efficacy of SP for the treatment of uncomplicated malaria in pregnant women. Countries that use SP for treatment or prevention of malaria in pregnancy need to evaluate their antenatal policy on timing or dose of FA supplementation.

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ABSTRACT
A Randomized Controlled Trial of Folate
Supplementation When Treating Malaria
in Pregnancy with Sulfadoxine-Pyrimethamine
Peter Ouma1, Monica E. Parise2, Mary J. Hamel3, Feiko O. ter Kuile4, Kephas Otieno1, John G. Ayisi1,
Piet A. Kager5, Richard W. Steketee6, Laurence Slutsker2, Anna M. van Eijk5*
1 Centre for Vector Biology and Control Research, Kenya Medical Research Institute, Kisumu, Kenya, 2 Division of Parasitic Diseases, National Center for
Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America, 3 Kenya Field Station, Centers for Disease
Control and Prevention, Kisumu, Kenya, 4 Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, United Kingdom, 5 Academic Medical
Centre, University of Amsterdam, Amsterdam, Netherlands, 6 Malaria Control and Evaluation Partnership in Africa, Program for Appropriate Technology
in Health, Batiment Avant Centre, Ferney-Voltaire, France
Trial Registration: NCT00130065
Funding: This study was funded by
CDC, USAID, and Dioraphte, a
private Dutch fund. The analytical
plan and the manuscript were not
influenced by the funding agencies.
The funders had no role in study
design, data collection and analysis,
decision to publish, or preparation
of the manuscript.
Competing Interests: The authors
have declared that no competing
interests exist.
Citation: Ouma P, Parise ME, Hamel
MJ, ter Kuile FO, Otieno K, et al.
(2006) A randomized controlled trial
of folate supplementation when
treating malaria in pregnancy with
sulfadoxine-pyrimethamine. PLoS
Clin Trials 1(6): e28. DOI: 10.1371/
journal.pctr.0010028
Received: June 2, 2006
Accepted: August 29, 2006
Published: October 20, 2006
DOI: 10.1371/journal.pctr.0010028
This is an open-access article
distributed under the terms of the
Creative Commons Public Domain
declaration which stipulates that,
once placed in the public domain,
this work may be freely reproduced,
distributed, transmitted, modified,
built upon, or otherwise used by
anyone for any lawful purpose.
Abbreviations: AHR, adjusted
hazard ratio; CI, confidence interval;
FA, folic acid; HR, hazard ratio; IPTp,
intermittent preventive treatment in
pregnancy; ITN, insecticide-treated
net; SP, sulfadoxine-pyrimethamine
* To whom correspondence should
be addressed. E-mail: amvaneijk@
yahoo.com
Objectives: Sulfadoxine-pyrimethamine (SP) is an antimalarial drug that acts on the folate
metabolism of the malaria parasite. We investigated whether folate (FA) supplementation in a
high or a low dose affects the efficacy of SP for the treatment of uncomplicated malaria in
pregnant women.
Design: This was a randomized, placebo-controlled, double-blind trial.
Setting: The trial was carried out at three hospitals in western Kenya.
Participants: The participants were 488 pregnant women presenting at their first antenatal
visit with uncomplicated malaria parasitaemia (density of ? 500 parasites/ll), a haemoglobin
level higher than 7 g/dl, a gestational age between 17 and 34 weeks, and no history of
antimalarial or FA use, or sulfa allergy. A total of 415 women completed the study.
Interventions: All participants received SP and iron supplementation. They were randomized
to the following arms: FA 5 mg, FA 0.4 mg, or FA placebo. After 14 days, all participants
continued with FA 5 mg daily as per national guidelines. Participants were followed at days 2, 3,
7, 14, 21, and 28 or until treatment failure.
Outcome Measures: The outcomes were SP failure rate and change in haemoglobin at day
14.
Results: The proportion of treatment failure at day 14 was 13.9% (19/137) in the placebo
group, 14.5% (20/138) in the FA 0.4 mg arm (adjusted hazard ratio [AHR], 1.07; 98.7%
confidence interval [CI], 0.48 to 2.37; p¼0.8), and 27.1% (38/140) in the FA 5 mg arm (AHR, 2.19;
98.7% CI, 1.09 to 4.40; p¼0.005). The haemoglobin levels at day 14 were not different relative
to placebo (mean difference for FA 5 mg, 0.17 g/dl; 98.7% CI,?0.19 to 0.52; and for FA 0.4 mg,
0.14 g/dl; 98.7% CI, ?0.21 to 0.49).
Conclusions: Concomitant use of 5 mg FA supplementation compromises the efficacy of SP
for the treatment of uncomplicated malaria in pregnant women. Countries that use SP for
treatment or prevention of malaria in pregnancy need to evaluate their antenatal policy on
timing or dose of FA supplementation.
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P PL Lo oS S CLINICAL TRIALS

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INTRODUCTION
In malaria endemic areas in sub-Saharan Africa, pregnant
women are more likely to be infected with Plasmodium
falciparum than nonpregnant women, affecting approximately
30 million pregnancies annually [1,2]. Adverse consequences
of malaria in pregnancy include maternal anaemia, maternal
mortality, low birth weight of the infant, and foetal loss [3,4].
The World Health Organization recommends three inter-
ventions for the control of malaria in pregnancy in areas of
stable transmission: intermittent preventive treatment, the
use of insecticide treated nets, and case management of
malarial illness and anaemia [5]. Many countries in sub-
Saharan Africa use sulfadoxine-pyrimethamine (SP) for the
treatment of clinical malaria in pregnancy or have intro-
duced intermittent preventive treatment in pregnancy (IPTp)
with SP as national policy [5]. IPTp consists of two or more
presumptive treatment doses of SP after the first trimester
delivered through the antenatal clinic, and has been shown to
reduce adverse effects of malaria in pregnancy [6–11]. Kenya
adopted this policy in 1998.
Folate (FA) supplementation in pregnancy has been
associated with reduction in anaemia and prevention of
megaloblastic erythropoiesis [12]; it is universally recommen-
ded as part of antenatal care. Although international guide-
lines recommend 0.4 or 0.6 mg of FA daily [13–15], many
countries in sub-Saharan Africa, including Kenya, use 5 mg
FA daily [16], because the 5 mg tablet is more widely available.
In areas of malaria transmission, IPTp with SP and FA are
often coadministered as part of antenatal care. However, the
mode of action of SP is based on the competitive inhibition of
two key enzymes in the biosynthesis of FA by the malaria
parasite. Several studies have shown that FA can antagonize
the antimalarial activity of SP in vitro and in vivo [17–21].
These studies, although not conducted among pregnant
women, have resulted in some public health authorities
recommending that FA should be temporarily withheld after
SP administration. However, temporary suspension of folate
makes program implementation complicated and may not be
necessary.
We conducted a randomized, double-blind, placebo-con-
trolled study among pregnant women with uncomplicated
malaria to assess whether FA 5 mg compromises the efficacy
of SP, and if a low dose of FA, such as 0.4 mg, may be an
acceptable alternative. The effect of maternal HIV infection
will be discussed in a separate manuscript.
METHODS
Participants
This study was conducted at three government hospitals in
western Kenya: Nyanza Provincial General Hospital in the
Kisumu District (population 500,000); Bondo District Hospi-
tal (district population 300,000), and Siaya District Hospital
(district population 480,000). In each site, HIV counselling
and testing is provided in the antenatal clinic as part of a
program to provide nevirapine to HIV-seropositive pregnant
women to reduce vertical transmission of HIV. Malaria
transmission is perennial and intense in western Kenya;
however, the malaria prevalence among pregnant women in
Kisumu is lower than in the rural areas of Bondo and Siaya.
Participants were recruited from the daily antenatal clinics in
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Folate and SP for Malaria in Pregnancy
Editorial Commentary
Background: Health authorities worldwide recommend that pregnant
women supplement their diet with folate (one of the B-vitamins),
normally 0.4 mg per day. There is good evidence from systematic
reviews of controlled trials that folate supplementation around
conception and early in pregnancy is effective in protecting against
neural tube (spine and brain) defects; continued supplementation
throughout pregnancy reduces the chance of anemia in the mother. In
many African countries, including Kenya, the dose of folate used is 5 mg
per day, because this dose is more easily available there. In Kenya, as well
as elsewhere in Africa, sulfadoxine-pyrimethamine is also given twice or
more after the first trimester to treat and/or prevent malaria infection
(which is more likely, and can have serious consequences, when a
woman is pregnant). However, there is some evidence from laboratory
experiments and clinical studies, none of which were done in pregnant
women, suggesting that folate supplementation might reduce the
effectiveness of sulfadoxine-pyrimethamine. Therefore, these researchers
conducted a trial to test this hypothesis in 415 pregnant Kenyan women
with malaria parasites in the blood but no severe symptoms. All were
given standard sulfadoxine-pyrimethamine treatment. The women were
randomized to receive either folate 5 mg daily, folate 0.4 mg daily, or
placebo tablets for 14 days, after which all women reverted to the
standard folate 5 mg tablets. The women were followed up for 28 days
after the initial sulfadoxine-pyrimethamine dose and the principal
outcome the researchers were interested in was the failure of
sulfadoxine-pyrimethamine treatment, defined as fever and the presence
of parasites in the blood (clinical failure) or the failure of parasites to clear
from the blood or to reappear too soon (parasitological failure).
What this trial shows: In this trial, women receiving folate 5 mg daily
were approximately twice as likely to fail treatment with sulfadoxine-
pyrimethamine than women receiving folate 0.4 mg or placebo. (Overall,
around 27% of the women receiving folate 5 mg had treatment failure
during the follow-up period.) All the treatment groups had similar levels
of blood hemoglobin at the end of the study. There did not seem to be
any major differences in adverse events (such as premature deliveries,
stillbirths, or neonatal deaths) among women taking part in the different
study groups.
Strengths and limitations: The randomization procedures were
appropriate and procedures were used to blind participants and
researchers to the different interventions, therefore reducing the risk of
bias. Since the trial had a placebo arm, it was possible to conclude that
the lower dose of folate (0.4 mg) did not significantly affect efficacy of
sulfadoxine-pyrimethamine as compared with placebo. A limitation of
the study is that the length of the intervention was short, since all
women reverted to standard 5 mg folate after 14 days. It is therefore not
clear whether a longer trial would have shown additional risks or
benefits of the different doses of folate. Finally, PCR genotyping was not
done on the parasites infecting women in the trial; this procedure could
have distinguished between true treatment failures and new infections
(but which would have been unlikely within 14 days).
Contribution to the evidence: Other trials and observational studies
have suggested that high doses of folate can reduce the efficacy of
sulfadoxine-pyrimethamine in children and adults. However these
studies have not examined the effect in pregnant women, for whom
most national bodies recommend regular folate supplementation. The
results from this trial supports the findings from previous studies and
enables the evidence to be generalized to pregnant women. The study
also found no evidence that 0.4 mg folate compromises the efficacy of
sulfadoxine-pyrimethamine. The findings suggest that the lower level of
folate dosing should be used in pregnancy, or that antimalarial
treatments other than sulfadoxine-pyrimethamine be used.
The Editorial Commentary is written by PLoS staff, based on the reports of the
academic editors and peer reviewers.

Page 3

the participating hospitals; the inclusion and exclusion
criteria are summarized in Table 1. The study protocol was
approved and reviewed on an annual basis by the institu-
tional review boards of the Kenya Medical Research Institute,
and the Centers for Disease Control and Prevention, Atlanta,
United States. All participants gave informed consent.
Interventions
A study nurse or clinical officer randomized participants to
FA 5 mg tablets (FA 5 mg arm), FA 0.4 mg tablets (FA 0.4 mg
arm), or placebo tablets (FA placebo arm); all were identical
in appearance and taste (Laboratory and Allied, Nairobi,
Kenya). Participants received a 14-day supply. At day 14, all
women received a supply of folic acid 5 mg tablets for 14 days
to ensure that pregnant women were not deprived of FA. The
first doses of FA or placebo were given together with SP
(three tablets of Malodar [Laboratory and Allied]: 1,500 mg of
sulfadoxine and 75 mg of pyrimethamine at once) under
supervision. Participants were observed for half an hour; if
vomiting occurred, the SP dose and FA tablet were repeated.
Participants were instructed to take the FA or placebo tablet
daily and were asked to bring the tablets at every visit for a
tablet count. All participants were supplemented with iron
tablets according to the national guidelines (200 mg three
times per day). From August 2004 onwards, all participants
received insecticide-treated nets (ITNs) as part of the enrol-
ment procedure to reduce the chance of new malaria
infections. Participants were instructed to return to the
clinic on days 2, 3, 7, 14, 21, and 28, or whenever they felt ill
and thought they needed treatment. On follow-up visits,
women were questioned about side effects, and signs and
symptoms of clinical malaria. The axillary temperature was
measured, and blood was obtained for a malaria blood smear;
haemoglobin was repeated on days 14 and 28. Women who
were ill or had complications that did not allow them to
continue participation were referred to the appropriate
departments in the hospital and followed until recovery.
Women who failed treatment with SP received quinine 600
mg three times per day for seven days. Women who had not
cleared parasitaemia after seven days of quinine therapy were
treated with mefloquine.
Haemoglobin was measured to the nearest 0.1 g/dl using a
portable haemoglobin monitor (HaemoCue, Mission Viejo,
California, United States). Peripheral thick and thin blood
films were stained with 10% Giemsa, and examined under oil
immersion for malaria parasites. A thick film was considered
negative if 100 microscopic fields showed no parasites.
Malaria parasites and leukocytes were counted in the same
fields until 300 leukocytes were counted. Parasite densities
were estimated by assuming a count of 8,000 leukocytes/ll of
blood. For quality control of the blood smear reading, 10% of
the negative samples and 20% of the positive samples at
screening, and 20% of all follow-up samples were checked by
a different microscopist during the study. HIV testing
involved parallel use of two rapid testing methods: Determine
HIV-1/2 (Abbott Laboratories, Dainabot, Tokyo, Japan) and
Unigold HIV-1/2 (Trinity Biotech, Bray, Ireland), as per Kenya
Ministry of Health guidelines for voluntary counselling and
testing. Capillus HIV-1/2 (Cambridge Diagnostics, Wicklow,
Ireland) was performed on discordant samples. The method
of Mount et al. [22] was used to test the urine for sulfa
compounds. The sickle cell profile was determined using
cellulose acetate electrophoresis (Helena Laboratories, Beau-
mont, Texas, United States).
Objectives
We investigated whether FA supplementation in a high or a
low dose affects the efficacy of SP for the treatment of
uncomplicated malaria in pregnant women.
Outcomes
Outcome measures were the prevalence of SP treatment
failure at days 3, 7, 14 (primary outcome), and 28 and change
in haemoglobin level comparing day 0 (day of SP treatment)
to days 14 and 28. Treatment failures were defined according
to the guidelines for an area of low to moderate transmission
(Table 2) [23]. The main difference from the protocol for
areas of high transmission is that in the moderate trans-
mission protocol an afebrile patient who still had para-
sitaemia on day 7 post-treatment was classified as a late
parasitological failure and given rescue treatment, whereas
such patients would not have been classified as parasitological
failures in the high-transmission protocol. Because of the
adverse consequences that asymptomatic parasitaemia can
.......................................................................................................................................................................................
Table 1. Inclusion and Exclusion Criteria
Inclusion Criteria Exclusion Criteria
Parasitaemia with a density of ? 500 parasites/ll (any species)
Gestational age 17–34 weeks
Willingness to provide blood samples and participate in HIV
counselling and testing
Haemoglobin . 7 g/dl
Available for the follow up period of four weeks
Informed consent
Aged 15–45 years
Use of FA in the last four weeks
Gestational age ? 16 wk or ? 35 weeks
History of an allergy to sulfa containing drugs or other unknown drugs
Haemoglobin ? 7 g/dl
An intake of sulfa containing drugs or 4-aminoquinolones in the previous month
A urine test positive for sulfa compounds
Sickle cell disease
Concomitant diseases needing treatment with cotrimoxazole or other sulfa-con-
taining drugs
Severe malaria or any other serious medical condition requiring hospitalisation or
additional treatmenta
aDanger signs or signs of severe malaria in adults are as follows. Clinical: prostration, impaired consciousness, respiratory distress, multiple convulsions, circulatory collapse, pulmonary
oedema (radiological), abnormal bleeding, jaundice, or hemoglobinuria; laboratory: severe anaemia (Hb , 7 g/dl), hypoglycemia, acidosis, hyperlactataemia, hyperparasitaemia, or
renal impairment [41].
DOI: 10.1371/journal.pctr.0010028.t001
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have for mother and foetus, we decided to use the more
conservative protocol.
Malaria was defined as the presence of asexual-stage
parasite of any species in thick smears, independent of
clinical signs. A parasite density in the highest tercile at
enrolment of the total study population was defined as a high
parasite density. A young age was defined as under 20 years.
Because of the short duration of follow-up and limited
sample size, no attempt was made to assess the effect of FA on
megaloblastic anaemia.
Sample Size
We calculated that a sample size of 600 women—200 in
each arm—would allow us to detect an increase from 5% to
15% in the parasitological failure rate at day 7 with 80%
power and 95% confidence, allowing for a 25% loss to follow-
up. However, we did not feel comfortable continuing the trial
at an overall treatment failure rate of over 40% at day 28 and
an intervention which may contribute to this, because it is
recommended that first-line therapy be changed at a 25%
failure rate [23,24]. An interim analysis was performed in
October 2005 with stopping criteria defined as a difference in
the treatment failure rate at day 14 with a p-value of less than
0.01; we used day 14 and not day 7 because we considered day
14 a more appropriate time point for assessing SP resistance.
Because this criterion was met, enrolment was stopped with
488 women enrolled. The investigators remained blind until
data cleaning, analysis, and quality control of the blood
smears were completed.
Randomization—Sequence Generation
One of the investigators generated a randomization list
with a block size of 12 using the statistical program SAS (SAS
system for Windows version 8; SAS, Cary, North Carolina,
United States).
Randomization—Allocation Concealment
All FA treatment and placebo tablets were prepared off site
and were identical in appearance and taste (Laboratory and
Allied). Medicine envelopes with 14 tablets of a treatment
arm were prepacked and labelled with the arm by staff who
were not involved in randomization. The medicine envelopes
were put in sealed, opaque envelopes with consecutive
numbers according to the randomization list by an inves-
tigator.
Randomization—Implementation
A trained clinical officer or nurse randomized eligible
women by assigning them the next envelope in order of
enrolment. The envelope was opened by the participant, and
the study arm was allocated by the study staff according to the
arm indicated on the medicine envelope.
Blinding
All FA treatment and protocol tablets were prepared off
site and were identical in appearance and taste (Laboratory
and Allied). All study staff participants were blind to the
treatment in each arm.
Statistical Methods
We analysed the data on an intention-to-treat basis using a
pre-established analysis plan. Cumulative treatment failures
by follow-up day were compared among treatment arms using
the Chi-squared test. We used the Kaplan-Meier curve to
examine differences in patterns between treatment arms, and
Cox proportional hazards regression analysis to examine the
effect of treatment arm on time to treatment failure after we
confirmed that the Cox proportional hazard assumption was
met. For day 14 post-treatment, we repeated the Cox
proportional hazards regression while adjusting for potential
confounders; factors examined included site of enrolment,
the use of an ITN, ethnicity, education level, socioeconomic
status, sickle cell status (carrier versus not a carrier), gravidity,
young age, HIV status, and a high parasite density at
enrolment. Analysis of covariance was used to assess the
effect of treatment arm on haemoglobin level [25]. Factors
were removed from models if the p-value was 0.05 or more.
The statistical program SAS (SAS system for Windows version
8) was used for all analyses. All tests were two-sided; p , 0.05
was considered significant, except for the efficacy between
study arms when a p , 0.013 was considered significant to
adjust for multiple comparisons and the interim analysis (the
.......................................................................................................................................................................................
Table 2. Definition of Treatment Failure
TermDescription
Early treatment failureDevelopment of danger signs or severe malariaaon days 1, 2, or 3 in the pres-
ence of parasitaemia
Parasitaemia on day 2 higher than day 0 count, irrespective of axillary tempera-
ture
Parasitaemia on day 3 with an axillary temperature ? 37.5 8C
Parasitaemia on day 3 ? 25% of count on day 0
Development of danger signs of severe malaria after day 3 in the presence of
parasitaemia without previously meeting any criteria of early treatment failure
Presence of parasitaemia and an axillary temperature ? 37.5 8C on any day from
days 4 to 28 without previously meeting any criteria of early treatment failure
Presence of parasitaemia on any day from days 7 to 28 and an axillary tempera-
ture , 37.5 8C without previously meeting any of the criteria of early treatment
failure or late clinical failure
Cumulative early, late clinical failure, and parasitological failure
Absence of early or late treatment failure on day 28
Late clinical failure
Late parasitological failure
Treatment failure
Adequate clinical and parasitological response
Definition of treatment failure based on recommendations of the World Health Organization [23].
aSee Table 1 for danger signs or signs of severe malaria in adults.
DOI: 10.1371/journal.pctr.0010028.t002
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p-value of 0.05 was subtracted by 0.01 to account for the
interim analysis, and the remaining value was divided by 3 to
account for the comparisons between the arms); a confidence
interval (CI) of 98.7% was used for the efficacy analysis.
RESULTS
Participant Flow
Between November 2003 and November 2005, a total of
4,524 women were screened; 488 met all enrolment criteria,
and 415 (85%) women completed the study (Figure 1). The
study arms were similar in baseline characteristics (Table 3).
Most infections were Plasmodium falciparum (98.0%), nine were
mixed P. falciparum/P. malariae, and one was pure P. malariae.
During 1,671 (99.4%) of the 1,682 routine visits made at or
before day 14, the participant reported that she took the FA
daily; the tablets were brought at 1,454 of the routine visits
(86.4%) and a correct count was established at 1,307 visits
(89.9%).
Outcomes and Estimation
From day 3 onwards, women in the FA 5 mg arm were more
likely to fail treatment than women in the other arms (Figure
2; log rank test p , 0.01 comparing the FA 0.4 mg arm or the
FA placebo arm to the FA 5 mg arm). On day 14 the number
of treatment failure was 38 out of 140 women (27.1%) in the
FA 5 mg arm, 20 out of 138 women (14.5%) in the FA 0.4 mg
arm, and 19 out of 137 women (13.9%) in the FA placebo arm
(Table 4). In multivariate analysis using Cox proportional
hazards regression, compared to FA placebo, treatment
failure by day 14 was twice as likely when FA 5 mg was used
(hazard ratio [HR], 2.19; 98.7% CI, 1.09 to 4.40; p ¼ 0.005),
whereas FA 0.4 mg did not affect treatment failure risk (HR,
1.07; 98.7% CI 0.48 to 2.37; p ¼ 0.8) (Table 5).
We did not find an effect of treatment arm on haemoglobin
levels at day 14 or day 28 among 288 women who completed
28 days of follow-up without treatment failure (Figure 3).
Among 386 women who had a haemoglobin available at day
14, the increases in mean haemoglobin in the FA 5 mg and FA
0.4 mg arms were not statistically different compared to the
FA placebo arm (0.17 g/dl; 98.7% CI,?0.19 to 0.52 g/dl; p¼0.3,
and 0.14 g/dl; 98.7% CI,?0.21 to 0.49 g/dl; p¼0.4, respectively;
adjusted for maternal HIV infection, location of residence,
high parasite density infection, and haemoglobin at enrol-
ment).
Figure 1. Trial Profile of the Study
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Adverse Events
During the course of the study, 20 participants (4.1%)
developed rashes (4, 8, and 8 in the FA 5 mg, FA 0.4 mg, and
FA placebo arms, respectively). No severe adverse skin
reactions or maternal deaths occurred. Premature delivery
was experienced by 14 participants (2.9%) (6, 5, and 3 in the
FA 5 mg, FA 0.4 mg, and FA placebo arms, respectively), and
eight participants (1.6%) had a stillbirth or early neonatal
infant death during the study (3, 2, and 3 in the FA 5 mg, FA
0.4 mg, and FA placebo arms, respectively).
DISCUSSION
Interpretation
This study shows that the combined use of SP and daily FA
supplementation in a dose of 5 mg compromised the efficacy
of SP for the treatment of malaria parasitaemia in pregnant
women. A plausible biological mechanism is available. FA is
required for DNA synthesis in both humans and protozoa.
Malaria parasites can utilize exogenous FA (the salvage
pathway) as well as synthesize FA de novo (biosynthesis)
[26], though biosynthesis seems to be the preferred method
[27]. Antifolates such as SP act on two enzymes important for
sequential steps in the biosynthesis of FA for the parasite,
dihydropteroate synthase and dihydrofolate reductase, re-
spectively. It has been established that malaria parasites can
differ in their ability to use exogenous FA, but the mechanism
is unknown [28,29]. If the biosynthesis pathway is compro-
mised, e.g., by sulfadoxine, parasite strains that are able to use
exogenous FA can compensate for the lack of FA through the
.......................................................................................................................................................................................
Table 3. Characteristics of Study Population at Enrolment, Overall and by Treatment Arm
CharacteristicDetailOverall, %
(n ¼ 488)
FA 5 mg, %
(n ¼ 161)
FA 0.4 mg, %
(n ¼ 165)
FA Placebo, %
(n ¼ 162)
Age
Gravidity
Trimester of pregnancy
Enrolment site
, 20 y
Primigravidae
Second
Kisumu
Bondo
Siaya
Luo
Married
None or incomplete primary
Primary complete
Secondary complete
House walls of mud
Possession of bicycle
Possession of ITNb
Received an ITN
Positiveb
Carrier
Any anaemia
Moderate anaemia
Documented feverd
Fever past week
High
Parasites/ll (95% CI)
50.8
52.5
69.1
41.6
24.0
34.4
91.4
63.9
45.7
46.1
8.2
48.6
83.4
15.0
62.7
34.1
20.3
86.1
13.5
3.7
58.6
33.4
3,231 (2,879 to 3,626)
47.8
52.8
68.9
41.6
26.1
32.3
93.2
62.7
45.3
47.8
6.8
50.3
82.6
13.0
63.4
38.5
21.1
87.0
14.3
4.4
55.9
35.4
3,178 (2,608 to 3,872)
52.1
58.2
71.5
39.4
24.9
35.8
94.6
61.2
41.8
46.7
11.5
46.1
82.4
16.5
61.8
28.7
21.2
89.1
13.9
3.0
56.4
30.3
3,149 (2,581 to 3,840)
52.5
46.3
66.7
43.8
21.0
35.2
86.4
67.9
50.0
43.8
6.2
49.4
85.2
15.5
63.0
35.2
18.5
82.1
12.4
3.7
63.6
34.6
3,373 (2,742 to 4,147)
Ethnic group
Marriage status
Education level
Indicator of socioeconomic statusa
ITN
HIV status
Sickle cell status
Anemiac
Fever
Parasite density
GMPD
aThe possession of a bicycle was used as an indicator of high/medium socioeconomic status. A house with mud walls in contrast to a house of bricks, walls of mud with cement, or
other materials was used as an indicator of low socioeconomic status.
bPossession of ITN, two missing; HIV status, indeterminate for one woman.
cAny anaemia: haemoglobin below 11 g/dl; moderate anaemia: haemoglobin below 8 g/dl.
dDocumented fever: An axillary temperature of 37.5 8C or higher.
GMPD, geometric mean parasite density
DOI: 10.371/journal.pctr.0010028.t003
.........................................................................................................................
Figure 2. Cumulative Treatment Survival Rates by Intervention Arm
among Parasitaemic Pregnant Women Treated with SP and FA
Participants received the FA intervention up to 14 days past SP treatment;
after day 14 every participant received FA 5 mg in accordance with the
National Guidelines in Kenya.
DOI: 10.1371/journal.pctr.0010028.g002
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Folate and SP for Malaria in Pregnancy

Page 7

biosynthesis pathway by increasing the flux through the FA
salvage pathway [27,28]. However, pyrimethamine may
interfere with the utilization of exogenous FA in a compet-
itive way, an action that is thought to be independent of
pyrimethamine’s inhibition of dihydrofolate reductase
[28,30]. The success and duration of the effect of pyrimeth-
amine may be dependent on the FA levels; large amounts of
FA (such as 5 mg daily), but not low doses, may overwhelm
pyrimethamine’s ability to block the salvage pathway [30].
It is likely that FA supplementation affects other antifolate
antimalarial combinations as well, such as chlorproguanil-
dapsone, dapsone-pyrimethamine, and cotrimoxazole. Cotri-
moxazole will increasingly be used as a prophylactic drug
among HIV-positive pregnant women. Further study into the
effect of concomitant FA supplementation in malarious areas
is needed.
We did not collect blood at enrolment and follow-up visits
to be able to differentiate between recrudescent and new
malaria infections. After day 14, all groups switched to FA 5
mg, so we were not able to assess the extent to which FA
contributes to SP treatment failure after 14 days. Several
studies indicate that FA supplements do not predispose to
increased risk of malaria acquisition [31,32], and thus we
hypothesized that the difference between treatment arms as
observed in this study after day 14 is mainly caused by
recrudescence.
Overall Evidence
Our results are supported by studies among symptomatic,
nonpregnant persons in areas of different malaria endemic-
ity. A randomized, placebo-controlled study in Gambia
reported approximately twice as common SP treatment
failures among children with symptomatic malaria supple-
mented with a high dose of FA (5 mg daily for children , 15
kg, 7.5 mg daily for children 15–20 kg, and 10 mg daily for
children . 20 kg) compared to the FA placebo group in an
area with low seasonal malaria transmission [19]. In a low-to-
moderate malaria transmission area in Kenya, a randomized,
open-label study among symptomatic participants (all ages)
showed a comparable cumulative survival curve when
assessing the interaction of SP and FA (5 mg daily) [20].
Dzinjalamala et al. [21] recently noted significantly higher
mean FA levels at enrolment among children with a
treatment failure to SP for symptomatic malaria (28 day
.......................................................................................................................................................................................
Table 4: Cumulative Treatment Failures and Relative Risk Reduction by Follow-Up Day among Participants Who Completed the
Study
Days Post-SP
Treatment
FA 5 mg,
n (%)
(n ¼ 140)
FA 0.4 mg
n (%)
(n ¼ 138)
FA Placebo,
n (%)
(n ¼ 137)
Total,
n (%)
(n ¼ 415)
Relative Risk Reduction
FA Placebo Versus FA
5 mg, % (98.7% CI), p-Value
Relative Risk Reduction
FA Placebo Versus FA
0.4 mg, % (98.7% CI), p-Value
Day 3
Day 7
Day 14
Day 28
14 (10.0)
21 (15.0)
38 (27.1)
78 (55.7)
5 (3.6)
12 (8.7)
20 (14.5)
49 (35.5)
5 (3.7)
12 (8.8)
19 (13.9)
51 (37.2)
24 (5.8)
45 (10.8)
77 (18.6)
178 (42.9)
63.5 (?28.0 to 89.6), p ¼ 0.06
41.7 (?35.9 to 74.9), p ¼ 0.16
48.9 (4.2 to 72.7), p ¼ 0.01
33.2 (6.9 to 52.1), p ¼ 0.003
?1 (?368.5 to 78.3), p ¼ 1.0
?1 (?164.5 to 61.6), p ¼ 1.0
4.3 (?99.5 to 54.1), p ¼ 1.0
?4.8 (?55.6 to 22.3), p ¼ 0.86
Participants received the intervention up to 14 days past treatment; after day 14 every participant received FA 5 mg in accordance with the National Guidelines in Kenya. A p-value
below 0.013 is considered significant to adjust for interim analysis and multiple comparisons.
DOI: 10.1371/journal.pctr.0010028.t004
......................................................
.......................................................................................
Table 5. The Effect of Daily FA Dose on Time to Treatment
Failure of SP among Pregnant Women by Follow-Up Time
Point
Day Ratio TypeHRs (98.7% CI)
FA Placebo FA 0.4 mgFA 5 mg
Day 3
Day 7
Day 14 Unadjusted
Day 28 Unadjusted
Day 14 Adjustedb
Unadjusted
Unadjusted
Reference
Reference
Reference
Reference
Reference
0.94 (0.20 to 4.50) 2.77 (0.76 to 10.06)
0.94 (0.34 to 2.60) 1.75 (0.71 to 4.31)
1.02 (0.46 to 2.27) 2.08a(1.04 to 4.18)
0.95 (0.58 to 1.56) 1.76a(1.12 to 2.74)
1.07 (0.48 to 2.37) 2.19a(1.09 to 4.40)
aSignificant HRs.
bAdjusted for young age and high-density parasitaemia.
DOI: 10.1371/journal.pctr.0010028.t005
...........................................................
Figure 3. Haemoglobin Levels by Intervention Arm at Different SP
Treatment Time Points
Haemoglobin levels (mean and 98.7% CI) are shown by type of FA
intervention at enrolment, 14 days, and 28 days post-treatment with SP for
malaria among 287 pregnant women who completed 28 days of follow-up
without treatment failure. Mean haemoglobin was obtained by analysis of
covariance and was adjusted for HIV, site of residence (rural versus urban),
and high parasite density. On days 14 and 28 the haemoglobin was adjusted
for haemoglobin at enrolment as well. Participants received the intervention
up to 14 days past SP treatment; after day 14 every participant received FA 5
mg in accordance with the National Guidelines in Kenya
DOI: 10.1371/journal.pctr.0010028.g003
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Folate and SP for Malaria in Pregnancy

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........................................................................................
follow-up) compared to children with an adequate para-
sitological and clinical response in Malawi.
Generalizability
SP is recommended for the treatment and prevention of
malaria in pregnancy. Although our results are based on the
treatment of uncomplicated malaria in pregnant women,
they will have implications for the use of SP as IPTp as well.
Many countries have introduced IPTp with SP [5]. We cannot
assess from our study the effect of FA 5 mg on the preventive
action of SP on malaria, but FA 5 mg will affect the treatment
action of SP when malaria parasitaemia is present. Depend-
ing on the endemicity of malaria in an area, it can be
expected that 1%–50% of pregnant women may carry
malaria parasitaemia, without noticing it, particularly in the
placenta [1,3]. Given the present results, countries using IPTp
should consider evaluating their FA recommendations in the
antenatal clinic to optimize SP efficacy. Options to consider
include using low-dose (0.4 mg) FA tablets daily or suspending
FA 5 mg for 14 days after SP treatment, which would disrupt
an important routine of daily intake of FA for the prevention
of anaemia. The first option may be preferable; our data show
no difference in efficacy of SP between 0.4 mg FA daily and
withholding FA 5 mg for 14 days. Effects of regimens on
haemoglobin levels were similar. However, this study was not
designed to assess the optimal FA dose to prevent FA
deficiency and adverse events such as megaloblastic anaemia
in the presence of malaria parasitaemia. International
guidelines recommend folic acid doses of 0.4 or 0.6 mg daily
during pregnancy [13–15]. Although these international
recommendations for FA supplementation are based on
studies conducted in developed countries, the few studies in
sub-Saharan Africa assessing FA deficiency among pregnant
women suggest that such deficiency is relatively uncommon,
ranging from 3%–10%; an exception was Togo (68% FA
deficiency among pregnant women) [33–38]. A dose of 1 mg
of FA daily in combination with malaria prophylaxis was
sufficient to abolish FA deficiency among primigravidae in
Zaria, Nigeria [39]. Given the international recommenda-
tions, the relatively low prevalence of FA deficiency in
pregnancy, and the compromised efficacy of SP for malaria
treatment when FA 5 mg is used, we believe it is reasonable to
recommend FA 0.4 mg daily for pregnant women in
malarious areas in sub-Saharan Africa.
Resistance to SP was high in the study area. However, at
present, no safe and efficacious alternative drug is available
for the treatment and prevention of malaria in pregnancy.
Kenya has moved now to artemisinin-based combination
therapy for children and quinine as first-line therapy for
clinical malaria in pregnancy, but IPTp with SP continues to
be used for prevention. A recent review of the efficacy of IPTp
with SP in the face of increasing SP resistance reported that in
areas with parasitological failure as high as 30% at day 14 in
children under 5 years of age, significant reductions in adverse
effects of malaria in pregnancy were seen when IPTp was used
[6–8]. However, alternatives for IPTp with SP urgently need to
be investigated. Considering the unique properties of SP in its
combination of treatment and prevention, including its low
cost and affordability, it may be worthwhile to preserve SP for
use as IPT among pregnant women or infants in areas where
SP resistance is low, and to use combination therapy with
other antimalarials for the treatment of symptomatic malaria,
reducing the drug pressure in the community treatment rate
[40]. Ensuring that the action of SP is not compromised by
concurrent high-dose FA supplementation will further in-
crease its therapeutic life.
SUPPORTING INFORMATION
CONSORT Checklist
Found at DOI: 10.1371/journal.pctr.0010028.sd001 (50 KB DOC).
Trial Protocol
Found at DOI: 10.1371/journal.pctr.0010028.sd002 (298 KB DOC).
ACKNOWLEDGMENTS
We thank Laboratory and Allied Limited in Nairobi, Kenya for
donating the study drugs (FA and SP for the participants). We thank
all the women who participated in this study for their patience and
understanding during the entire consenting process, and the enrol-
ment and follow-up period. Special thanks to our study staff and the
medical staff of PGH, Siaya, and Bondo hospitals. We would like to
thank the director of Kenya Medical Research institute for his
support and permission to publish this paper, and John Williamson
for his statistical advise.
Author Contributions
MEP, FOtK, JGA, LS, and AMvE designed the study. PO,
JGA, and AMvE analyzed the data. PO and AMvE enrolled
patients. PO, MEP, MJH, FOtK, KO, JGA, PAK, RWS, LS, and
AMvE wrote the paper. PO supervised field work and
coordinated all field activities. MJH provided supervision to
the study team in the field, backstopping and providing
support to PO, who was primarily responsible for the team on
the ground in Kenya, and provided input into the inter-
pretation of data. KO carried out and supervised the
laboratory procedures. RWS provided initial input into the
design of the study along with the lead author and other
authors and provided input on drafts of the manuscript as it
was developed.
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