Sulfadoxine-Pyrimethamine–Based Combinations for
Malaria: A Randomised Blinded Trial to Compare Efficacy,
Safety and Selection of Resistance in Malawi
David J. Bell1,5*, Suzgo K. Nyirongo2, Mavuto Mukaka2, Ed E. Zijlstra3, Christopher V. Plowe4, Malcolm E.
Molyneux2, Steve A. Ward1, Peter A. Winstanley5
1Department of Molecular and Biochemical Parasitology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom, 2Malawi-Liverpool-Wellcome Trust Clinical
Research Programme, Blantyre, Malawi, 3Department of Medicine, College of Medicine, University of Malawi, Blantyre, Malawi, 4Center for Vaccine Development,
University of Maryland School of Medicine, Baltimore, Maryland, United States of America, 5School of Clinical Sciences, University of Liverpool, Liverpool, United Kingdom
Background: In Malawi, there has been a return of Plasmodium falciparum sensitivity to chloroquine (CQ) since sulfadoxine-
pyrimethamine (SP) replaced CQ as first line treatment for uncomplicated malaria. When used for prophylaxis, Amodiaquine
(AQ) was associated with agranulocytosis but is considered safe for treatment and is increasingly being used in Africa. Here
we compare the efficacy, safety and selection of resistance using SP or CQ+SP or artesunate (ART)+SP or AQ+SP for the
treatment of uncomplicated falciparum malaria.
Methodology and Findings: 455 children aged 1–5 years were recruited into a double-blinded randomised trial comparing
SP to the three combination therapies. Using intention to treat analysis with missing outcomes treated as successes, and
without adjustment to distinguish recrudescence from new infections, the day 28 adequate clinical and parasitological
response (ACPR) rate for SP was 25%, inferior to each of the three combination therapies (p,0.001). AQ+SP had an ACPR
rate of 97%, higher than CQ+SP (81%) and ART+SP (70%), p,0.001. Nineteen children developed a neutropenia of
#0.56103cells/ml by day 14, more commonly after AQ+SP (p=0.03). The mutation pfcrt 76T, associated with CQ resistance,
was detected in none of the pre-treatment or post-treatment parasites. The prevalence of the pfmdr1 86Y mutation was
higher after treatment with AQ+SP than after SP, p=0.002.
Conclusions: The combination AQ+SP was highly efficacious, despite the low efficacy of SP alone; however, we found
evidence that AQ may exert selective pressure for resistance associated mutations many weeks after treatment. This study
confirms the return of CQ sensitivity in Malawi and importantly, shows no evidence of the re-emergence of pfcrt 76T after
treatment with CQ or AQ. Given the safety record of AQ when used as a prophylaxis, our observations of marked falls in
neutrophil counts in the AQ+SP group requires further scrutiny.
Trial Registration: Controlled-Trials.com ISRCTN22075368
Citation: Bell DJ, Nyirongo SK, Mukaka M, Zijlstra EE, Plowe CV, et al (2008) Sulfadoxine-Pyrimethamine–Based Combinations for Malaria: A Randomised Blinded
Trial to Compare Efficacy, Safety and Selection of Resistance in Malawi. PLoS ONE 3(2): e1578. doi:10.1371/journal.pone.0001578
Editor: Aric Gregson, University of California Los Angeles, United States of America
Received September 10, 2007; Accepted January 9, 2008; Published February 13, 2008
Copyright: ? 2008 Bell et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by a Wellcome Trust Training Fellowship in Clinical Tropical Medicine awarded to David Bell (grant number 066681). The funders
had no role in the study design or execution, or in the analysis and presentation of the data.
Competing Interests: Peter Winstanley and Steve Ward are unpaid members of the MMV-GSK product development team for chlorproguanil-dapsone-
artesunate. None of the other authors declare any conflicts of interest.
Malaria isresponsible for around 1 million deaths annually insub-
Saharan Africa, especially in young children. Attempts to control
malaria have been hampered because of resistance in Plasmodium
falciparum to the most commonly used drugs, chloroquine (CQ) and
sulfadoxine-pyrimethamine (SP).MalawiswitchedfromCQto SPas
first line treatment for uncomplicated falciparum malaria in 1993,
the first country in sub Saharan Africa to do so. SP failure rates have
risen since, to day 28 parasitological failure rates of 73% in 2002 
and 79% in 2005 . In the light of this declining efficacy, Malawi is
changing its first line therapy again. The World HealthOrganisation
(WHO) recommends the use of combination therapies of two or
more drugs that target different pathways to overcome resistance-in
particular, artemisinin combination therapies (ACTs) . Many
countries in sub Saharan Africa have adopted ACTs though in
several of these countries, this policy has yet to be implemented .
The combination amodiaquine (AQ) plus SP has been shown in
some African countries to have efficacy similar to ACTs . AQ is a
4-aminoquinoline like CQ but remains efficacious in many areas
with substantial CQ resistance [6,7]. When taken weekly to prevent
malaria, amodiaquine was associated with agranulocytosis and
lacking, but its safety is considered to be acceptable . AQ is
increasingly being used in Africa, usually in combination with
artesunate (ART). Chloroquine (CQ) plus SP has been less
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efficacious than AQ+SP in trials in Africa owing to widespread CQ
resistance. CQ-resistant parasites carry the 76T mutation on the
gene pfcrt . In Malawi, the prevalence of this marker has fallen
since the switch from CQ to SP (85% in 1992, 0% in 2001) . In
addition there has been a fall in the prevalence of mutations in the
gene pfmdr1; the association of these mutations with CQ resistance is
less clear. Recently a return of in vivo CQ sensitivity, predicted by
these molecular changes, has been reported from Malawi .
Here we report the results of a randomised double-blind clinical
trial comparing the efficacy and safety of 3 SP based combination
therapies, CQ+SP, artesunate (ART)+SP and AQ+SP with that of
SP alone for the treatment of uncomplicated malaria in young
children in Malawi. At the time of the study, SP monotherapy was
the treatment recommended for uncomplicated malaria by the
Malawi National Malaria control committee. In addition, we
compared the selection of resistance associated mutations in the
parasite genes dhfr and dhps (associated with SP resistance ) and
pfcrt and pfmdr1 (associated with AQ and CQ resistance).
The protocol for this trial and supporting CONSORT checklist
are available as supporting information; see Checklist S1 and
The study was based at Chileka health centre near Blantyre,
Malawi, where malaria transmission is perennial, peaking during
December to April. Between September 2003 and December
2005, children presenting with an illness suggesting falciparum
malaria were screened. Inclusion criteria were: i) age $12 and
,60 months, ii) weight $6 kg, iii) axillary temperature $37.5uC,
iv) no history of treatment with an antimalarial, cotrimoxazole or a
tetracycline antibiotic in the previous week, v) no features
suggesting severe malaria or a concomitant illness, vi) haemoglobin
$5.0 g/dl using HemocueH, and vii) P. falciparum monoinfection
with a parasite density between 2000 and 200,000 parasites per ml.
Written informed consent was required from the parent of each
child recruited. The study protocol was approved by ethics
committees of the College of Medicine, University of Malawi and
Liverpool School of Tropical Medicine. A data and safety
monitoring board and local study monitor were appointed.
Treatment, Randomisation and Blinding
Children meeting all inclusion criteria on day 0 were recruited
and randomised to one of four treatment groups. Randomisation
was in blocks of 12 according to an off-site computer-generated
code to assign patients equally to the four oral treatment groups:
SP (25 mg/kg sulfadoxine and 1.25 mg/kg pyrimethamine as a
single dose on day 0)+vitamin C 50 mg tablet (placebo) daily for
3 days; CQ (10 mg/kg on days 0 and 1, and 5 mg/kg on day
2)+SP; ART (4 mg/kg once daily for 3 days)+SP; or AQ (10 mg/
kg daily for 3 days)+SP. In the case of children too young to
swallow tablets, CQ syrup (50 mg per 5mls) and AQ syrup (50 mg
per 5mls) were used (same doses as above). The other study drugs
were not available as syrups and were crushed and given on a
spoon with water if the child could not swallow a tablet. The
different tablets were not identical in appearance or taste. A three-
day supply of paracetamol (10 mg/kg) was given.
Each child was given a unique study number, assigned
sequentially. A dedicated study ‘drug dispenser’ opened the
corresponding randomisation envelope and directly observed all
drug doses but was not involved in the assessment of children. All
other members of the study team were blinded to the dispensing
process and patients were uninformed of their treatment allocation
for the duration of the study. Children were observed for
30 minutes after dosing. If the child vomited, a second dose was
given. If vomiting occurred a second time, the child was
withdrawn and treated with parenteral quinine.
Classification of Outcomes
Patients were assessed on days 0, 1, 2, 3, 7, 14, 28 and 42 and
any other day if unwell. Blood was collected for parasite
microscopy, storage on Whatman 3M filter paper and, at specified
visits, for determination of the full blood count and biochemical
parameters. Clinical outcome was assessed using the 2003 WHO
therapeutic efficacy protocol for areas of intense malaria
transmission . Participants were withdrawn if they failed to
attend for follow up, withdrew consent or took a ‘banned’ drug i.e.
all antimalarials, cotrimoxazole, doxycycline, tetracycline, chlor-
pheniramine and folic acid. Late clinical failures were treated with
oral mefloquine (25 mg/kg). Severe malaria was treated with
parenteral quinine in hospital.
The planned sample size of 100 evaluable patients per
treatment arm was calculated to have 90% power to detect the
difference between an ‘‘adequate clinical and parasitological
response’’ (ACPR) rate of 80% with SP alone and 95% with
combination therapies using the 5% significance level for each
comparison with SP alone. The primary endpoint was the day 28
ACPR rate and the major analysis strategy for the primary
endpoint was intention to treat (ITT). Patients with missing
outcomes were all classified as successes in one analysis and then as
failures in a separate analysis. Per protocol (PP) analysis was also
done using polymerase chain reaction (PCR) corrected data to
distinguish recrudescences from reinfections. When PCR analysis
indicated that a post-treatment parasitaemia was a reinfection, the
outcome was classified as a treatment success on that day, but
excluded from subsequent analyses. If PCR was inconclusive, the
case was excluded from the analysis.
Secondary endpoints included day 14 and 42 ACPR rates, time
to fever resolution (axillary temperature#37.5uC), time to parasite
clearance, change in haemoglobin from day 0 to day 14 and the
appearance of gametocytes by day 28 after treatment. We also
compared adverse events (AEs) between the treatment groups,
including self-reported AEs and laboratory AEs; rises in alanine
transferase (ALT), total bilirubin, and creatinine between days 0
Data were double entered and validated prior to the analyses.
Data analysis was performed using Stata 8. Binomial regression
was used to obtain risk differences between treatments and 95%
confidence intervals. Fisher’s exact p-values were reported. Tests
of significance were performed using the 5% level to infer
significance for the planned analyses. Pair wise comparisons
between combination therapies were not planned and in these
comparisons we adjusted the significance level to 1.7% (i.e.
p,0.017) using Bonferroni’s approach.
To look for evidence of selection of resistance mutations in the
genes dhfr, dhps, pfcrt and pfmdr1, we compared the prevalence of
resistance mutations in these genes before and after treatment,
both within each of the four different treatment groups and
between the four treatment groups.
Blood films were stained with Fields stain and parasite densities
estimated from thick films by counting the number of parasites per
200 white blood cells (WBC) assuming a total count of 8000/ml.
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These parasite counts and haemoglobin (Hb) estimates using
HemocueH were used for screening purposes. In addition, on days
0 and 14, the full blood count was measured using a Beckman
Coulter HMX and plasma ALT, total bilirubin and creatinine
using a Vitros DTII dry biochemistry analyser. The actual WBC
count from the coulter was subsequently used to calculate an
accurate parasite count for the analyses. The presence or absence
of gametocytes was noted on each blood film.
Parasite DNA was extracted from dried blood on filter paper. A
nested PCR was used to distinguish recrudescent infections from
new infections in all patients with parasitaemias appearing from
day 12 onwards. The msp2 gene was amplified and size
polymorphisms identified by gel electrophoresis using previously
described methods . Parasites were classified as recrudescent if
they shared any of the bands that were present on day 0 and as
reinfections if they had no bands in common. Parasites that
appeared before day 12, having initially disappeared, were
assumed to be recrudescent. Nested PCR followed by mutation-
specific restriction enzyme digestion was used to determine the
prevalence of different alleles in the dhfr, dhps, pfcrt and pfmdr1 genes
in day 0 parasites and parasites appearing at any time from day 12
onwards after treatment. A detailed description of these techniques
is available at http://www.medschool.umaryland.edu/cvd/plowe.
asp. We analysed the dhfr gene for polymorphisms at codons 51,
59, 108 and 164; dhps codons 437, 540 and 581; pfcrt codon 76;
and pfmdr1 codons 86 and 1246. Samples were analysed in a
blinded fashion with respect to the treatment groups.
Recruitment and Participant flow
We screened 1625 children and 455 met all inclusion criteria and
were enrolled. Baseline characteristics are shown in table 1 and the
study profile in figure 1. By day 14, 44 (9.7%) children had been
withdrawn from the study, 51 (11.2%) by day 28 and 63 (13.8%) by
day 42. The reasons for withdrawal are summarised in figure 1.
Primary Outcome: Treatment Efficacy
Using the ITT approach with missing outcomes treated as
successes, the day 28 ACPR rate was lowest with SP alone at 25%
and inferior to each of the three SP combination therapies
(p,0.001), table 2. AQ+SP had an ACPR rate of 97%, higher
than each of CQ+SP and ART+SP (p,0.001). There was no
significant difference between CQ+SP and ART+SP.
156 recurrent parasitaemias occurred 12 or more days after
treatment. PCR analysis showed that 97 (62%) were recrudes-
cences, 56 (36%) were reinfections and for 3 (2%), the analysis
failed. Figure 2 shows the proportions of these in the four
treatment groups (recurrent parasitaemias before 12 days were
assumed to be due to recrudescence). The PP analysis of these
PCR corrected data showed that treatment with SP was inferior to
all the SP combinations on day 14, 28 and 42 (p,0.001), figure 3.
On day 28, AQ+SP was more efficacious than CQ+SP (p=0.009)
and ART+SP (p,0.001) and on day 42, more efficacious than
ART+SP (p=0.004). The difference between AQ+SP and
CQ+SP was not statistically significant on day 42 (p=0.03) after
adjustment using Bonferroni’s approach.
Ninety-five percent of children had cleared their parasite by day
2 in the ART+SP group compared to 35% for SP, 47% for
CQ+SP, and 55% for AQ+SP (p,0.001 for each comparison with
AQ+SP). By days 3 and 7, there were no differences between the
three combination therapies and they were all superior to SP
alone, p=0.005. In the SP group, there was no association
between the day 0 parasitaemia and time to parasite clearance or
between day 0 parasitaemia and clinical outcome. Fever resolution
was slower with SP alone; the percentage of children who still had
fever on day 1 were 18% for SP, 5% for CQ+SP, 6% for ART+SP
and 5% for AQ+SP (p,0.008 for each comparison with SP).
Mean haemoglobin concentration rose in all treatment groups.
Compared to SP alone, the adjusted mean on day 14 was greater
Table 1. Summary of Baseline characteristics by treatment group
Number of patients114113114 114
Median age (months), (IQR)22.6 (17.6)22.2 (14.0)21.6 (8.2) 21.0 (14.2)
Number (%) female62 (54.4%) 54 (47.8%) 51 (44.7%)56 (49.1%)
Number of days of fever 3.1 (1.8)2.9 (1.8) 3.1 (1.9) 3.1 (1.7)
Weight (kg)10.9 (2.2) 10.8 (2.3)10.8 (2.1)10.8 (2.4)
Initial temperature (uC)
Geometric mean (range) parasite count* (per ml)
Parasite count.200,000/ml (%)
38.8 (0.9)38.7 (0.9)38.8 (0.9) 38.8 (0.9)
66,171 (973–301,294) 59,874 (2146–280,720)36,315 (1077–306,722)44,356 (1892–288,353)
10 (8.8%) 16 (14.2%)9 (7.9%)13 (11.4%)
Gametocytes seen on Day 0 18 (15.8%)15 (13.3%)18 (15.8%)19 (16.7%)
Haemoglobin** (g/dl)9.1 (1.7)9.0 (1.6)8.9 (1.4)9.3 (1.6)
White cell count (x109/L) 10.2 (4.3) 10.8 (4.6)9.9 (4.4)10.3 (4.7)
Neutrophil count (x109/L) 5.1 (3.6)5.2 (3.6) 4.1 (2.9)5.3 (4.1)
Platelet count (x109/L) 149 (91)165 (93) 163 (100)162 (103)
Alanine Transferase (IU/L)19 (13) 18 (17)18 (16) 22 (19)
Total Bilirubin (mg/dl) 0.97 (0.5)0.95 (0.7) 0.88 (0.7)0.85 (0.6)
Creatinine (mg/dl)0.4 (0.1)0.4 (0.1)0.4 (0.1)0.4 (0.1)
For all, data shows mean (SD) unless otherwise indicated
*Parasite count calculated using coulter HB and WCC
**HB from coulter counter. If not available, the Hemocue HB was used (n=5)
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Figure 1. Study Profile
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after CQ+SP (p=0.03) and AQ+SP (p=0.002) but not after
ART+SP (p=0.81). Gametocytes were present on day 0 in 73 (16%)
children, table 1. There were no differences between the groups in
the percentage of children with gametocytes on day 28; 4% after SP,
7% after CQ+SP, 5% after ART+SP and 7% after AQ+SP.
Safety and Tolerability
284 clinical AEs were reported in 185 children. Cough was
commonest, making up 45% of all AEs. Compared to SP alone,
cough was more commonly reported after ART+SP, p=0.04. No
other statistically significant differences were found. There were 8
serious adverse events (SAEs) in the study with no more than 4 in
any treatment group. There were no deaths. The SAEs included 2
cases of pneumonia requiring intravenous antibiotics, 1 child with
gastroenteritis requiring intravenous fluids and 5 treatment failures
requiring hospitalisation for intravenous quinine. For two of these
children, this was due to the occurrence of seizures shortly after
receiving their medication on day 0 (1 AQ+SP, 1 CQ+SP), and 1
child had persisted vomiting on day 1 and was unable to continue
oral treatment (AQ+SP). The other two children had early
treatment failures (1 SP, 1 AQ+SP), and were too unwell to
continue oral therapy.
Neutrophil counts fell after treatment in all treatment groups
but the proportion of children with neutrophil counts of
#0.56103/ml on day 14 (having been $1.06103/ml on recruit-
ment) was greatest in the AQ+SP group (11.5%, n=7), higher
than the SP alone group (1.5%, n=1), p=0.03. For 17 of the 19
children in which this low neutrophil count was observed, a repeat
sample between day 28 and 42 showed neutrophils $1.06103/ml.
For the remaining 2 children no further sample was obtained. We
noted no evidence of any ill effects due to these transient low
Two children in the AQ+SP treatment group developed plasma
ALT levels on day 14 greater than three times the upper limit of
normal having been in the normal range (15–45 U/L) on
recruitment. The possible association of this adverse effect with
AQ+SP was not significant, p=0.24. One of these children had a
day 14 ALT of 1540 U/L with a total bilirubin of 1.1 mg/dl (0.1–
1.4 mg/dl). By day 42 the ALT had returned to normal. The
other child had a day 14 ALT of 473 U/L with a total bilirubin of
2.2 mg/dl. No further samples were collected for analysis. Both
children were well and completed 42 days of follow up and both
had day 14 neutrophil counts $1.06103/ml.
Selection of Resistance
The resistance mutations pfcrt 76T, dhfr 164L and dhps 581G were
detected in none of the pre-treatment (n=244, 155 and 134
respectively) or post-treatment (n=151,145 and 95) infections.
Pfmdr1 86N (wild type) parasites were present in 219 out of 244
(89.8%) of pre-treatment infections and in 88.1% (134 out of 152)
post-treatment infections. Within each treatment group, there was
after treatment. When comparing between treatment groups, the
proportions of pfmdr1 86Y (mutant) in the post treatment AQ+SP
group (in 4 recrudescences and 1 new infection) was higher than in
the SP post-treatment group, p=0.002. Pfmdr1 1246D (wild type)
parasites were present in 224 out of 231 (97%) of pre-treatment
infectionsand in94.5% (86 out of 91) post-treatment infections. This
difference was not significant, and there were no differences within
or between the four different treatment groups.
The prevalence of dhfr mutant alleles was very high in pre-
treatment parasites; 99% (156/157) were 108N, 99.5% (254/255)
were 51I and 96% (357/371) were 59R. Dhps mutants were also
common pre-treatment; 97% (33/34) were 437G and 95.1%
(355/373) were 540E. Pre-treatment, 91.6% (339 out of 370) of
parasites were ‘‘quintuple’’ mutants (all mutant alleles at dhfr
codons 51, 59 and 108 and dhps codons 437 and 540) and post-
Table 2. Day 28 ACPR rates and differences (95% confidence intervals) between the groups by intention to treat analysis–data not
Comparison ITT analysis (Withdrawals counted as successes) ITT analysis (Withdrawals counted as failures)
confidence interval)P-valueSuccess rate
CQ+SP vs. ART+SP
CQ+SP vs. AQ+SP
ART+SP vs. AQ+SP
81%56% (45%, 67%)
,0.00167% 48% (37%, 59%)
70%45% (33%, 56%)
,0.001 60%40% (29%, 52%)
97% 72% (63%, 80%)
,0.00184%64% (54%, 74%)
211% (222%, 20.2%) 0.063
28% (220%, 5%) 0.271
16% (8%, 24%)
,0.00116% (5%, 27%) 0.006
27% (18%, 36%)
,0.00124% (12%, 35%)
Figure 2. Numbers of recrudescent and new infections in those
children with recurrent parasitaemia after treatment.
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treatment, this figure was 92% (139/151). Paired analysis of
parasites, pre and post treatment, showed no significant differences
within or between the treatment groups.
Ten years after the introduction of SP as first line treatment for
uncomplicated malaria in Malawi the day 28 ACPR rate lies below
30%. Over 90% of pre-treatment parasites were ‘‘quintuple’’
mutants; this genotype has been shown to be strongly predictive of
SP failure in young children in Malawi . Malawi is in the process
and our data support the decision to abandon SP. This artemisinin-
an adult course. However, it is a relatively complex regime, 6 doses
over 3 days with food, and until recently in Africa, supplies of the
drug have not matched the huge demand.
Low cost alternatives to artemether-lumefantrine might have
been attractive to the Malawi authorities and AQ+SP was one of
the combinations considered. In most countries in Africa,
antimalarials are obtained without prescription and often taken
unnecessarily . In the era of ACTs, such unregulated use will
be hard to sustain. Four other fixed ratio ACTs are in
development, AQ plus ART is now available in Africa,
chlorproguanil-dapsone plus ART has finished phase III testing,
and will be submitted to the regulatory authorities in 2008, and
piperaquine plus dihyroartemisinin and pyronaridine plus ART
are well into phase III trials. Each of these is likely to be cheaper
and more practicable to use than artemether-lumefantrine but still
likely to be more expensive that AQ+SP or CQ+SP.
This study confirms the return of CQ sensitivity in Malawi and
importantly, shows no evidence of the re-emergence of pfcrt 76T
after treatment with CQ or AQ based combinations. The CQ+SP
day 28 PP ACPR rate was 86%, lower than the rate of 99%
reported from another randomised study in 2005, also in Blantyre,
in which CQ was used as a monotherapy in 80 children with
uncomplicated malaria . The children in our study were
younger, mean age 22.2 vs. 31.2 months, and had higher parasite
counts on enrolment, and this may explain some of this difference.
Another possible reason, especially in the absence of pfcrt 76T
mutants, is that some children in this study may have failed to
achieve therapeutic CQ concentrations. A pharmacokinetic (PK)
study was nested within this study and data on drug levels will be
AQ+SP was significantly more efficacious than CQ+SP,
ART+SP and SP alone. AQ+SP also appears to have a longer
period of post-treatment prophylaxis than the other treatments;
reinfection rates on days 14, 28 and 42 were similar after
treatment with SP, CQ+SP or ART+SP but with AQ+SP, no
reinfections were seen until after day 28 (figure 2). AQ+SP was
more efficacious than CQ+SP against a background of 100% pfcrt
76 wild type parasites. In vitro, AQ has greater activity against P.
falciparum than CQ , and this greater potency may be evident
here. Alternatively, the lower CQ+SP efficacy may have PK basis
and data from the PK study will be used to address this. Treatment
with AQ+SP was associated with the selection of parasites with the
pfmdr1 86Y mutation between day 28 and day 42 after treatment.
The combination of ART+SP for 3 days was the least effective
of the three combination therapies. This is not surprising given the
poor efficacy of SP. As a monotherapy, ART is usually taken for
7 days; a 3-day ACT course is only efficacious when the second
drug retains adequate efficacy. The combination of ART+SP has
proven ineffective in other sites in Africa with significant
background SP resistance [19,20]. This study highlights the
Figure 3. ACPR rates by per protocol analysis, using PCR corrected data.
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importance of continuing follow up beyond 14 days; ART+SP had
a 93% day 14 ACPR rate and had the fastest parasite clearance
rate but efficacy by days 28 and 42 was poor.
Generalizability and Overall evidence
AQ+SP has shown excellent efficacy in several African studies
and a recent meta-analysis concluded that the efficacy of AQ+SP
in Africa was similar to that of AQ+ART but inferior to
artemether-lumefantrine .The results of these studies showed
considerable variability, in part due to differences in existing
background resistance to SP and AQ and also to differences in the
transmission intensities at the study sites; AQ+SP was more
efficacious in high transmission areas. This is probably due to
increased host acquired immunity in these areas and also to the
post-treatment prophylactic properties of the combination. AQ is a
pro-drug, but its active metabolite desethyl AQ has a half-life of
several weeks, similar to chloroquine and sulfadoxine. However,
post-treatment prophylaxis comes at a cost; drugs with long
terminal elimination phases promote the selection of parasites with
resistance-conferring mutations . As the drug concentration
falls to sub-therapeutic levels, recrudescent or newly infecting
parasites carrying resistance mutations may be preferentially
selected. This is especially important in areas of moderate to high
transmission where re-infections are common and parasite
densities may be high.
We saw evidence of the selection of the pfmdr1 86Y mutation
after treatment with AQ+SP. This mutation is associated with in
vivo AQ resistance . Other studies have demonstrated the
selection of pfmdr1 86Y and pfmdr1 1246Y mutations after
treatment with AQ+ART [23,24], and after treatment with
AQ+SP . Studies from Zanzibar and Uganda have demon-
strated significant increases in parasites carrying the pfmdr1 86N
allele after treatment with artemether-lumefantrine [26,27]. This
allele has been associated with decreased lumefantrine sensitivity
in vitro . These studies provide evidence that these
combination therapies are able to drive the selection of resistance
mutations and it will be important to monitor closely their efficacy,
especially in high transmission areas, to see whether these
genotypic changes translate into treatment failures. Artemisinins
have short half lives (measured in hours) while all of the
companion drugs (apart from chlorproguanil-dapsone) in ACTs
recommend by the WHO or under development have half-lives in
the order of weeks. Whether the combination chlorproguanil-
dapsone plus ART, in which both component drugs have short
(closer matched) half-lives, will prevent this selection process,
remains to be seen.
We were unable to detect dhfr 164L mutants using conventional
PCR. This mutation is associated with failure of chlorproguanil-
dapsone. One group, using real time PCR, has reported this
mutation in 4 of 85 (4.7%) samples from pregnant women in
Blantyre collected in 2003 . We were unable to detect any dhfr
164L mutants in 158 samples collected in this study using a
modified version of the same method, suggesting that if the
mutation is present in this region, it remains extremely rare
(Ochong E et al, in preparation).
AQ was withdrawn as a prophylaxis against malaria in 1986,
because of agranulocytosis (1 in 2,100 subjects) and hepatitis (1 in
15,650) . These AEs have not been reported when AQ has
been taken as malaria treatment. Between 1984 and 1987, 20
cases of AQ associated agranulocytosis were reported in the
literature in individuals taking AQ for malaria prophylaxis
[8,9,31–34]. The shortest duration of prophylaxis prior to
diagnosis of agranulocytosis was 3 weeks and lowest total dose
consumed 1.2 grams. The treatment dose for a 70 kg adult is 2.1
grams. Neutrophil counts fell after treatment in all treatment
groups in this study but the proportion of children with neutrophil
counts of #0.56103/ml on day 14 was higher in the AQ+SP group
than the SP alone group, p=0.03. We also observed marked rises
in ALT in 2 children after treatment with AQ+SP though this
association was not found to be significant. These observations
involve small numbers of children and the study was not powered
to detect rare AEs. We do however think that given the track
record of safety issues with AQ, these observations require further
scrutiny especially as AQ may be used increasingly in African
patients who have several episodes of malaria each year.
SP has failed in Malawi after a decade of useful service, and its
role in intermittent presumptive therapy in pregnancy (IPT)
should also now be re-evaluated. There has been a return of CQ
sensitivity in Malawi, and CQ could be considered as a possible
replacement for SP in IPT programmes. The potential for CQ to
be used as part of combination therapy should be considered with
caution unless and until CQ-sensitive parasites predominate
throughout the region. Given the extensive cross-border move-
ment of people, it seems likely that CQ-resistant parasites from
neighbouring countries would be selected rapidly on redeployment
of this drug.
The combination AQ+SP was highly efficacious, despite the low
efficacy of SP alone; however, we found evidence that in these
circumstances AQ may exert selective pressure for resistance
mutations many weeks after treatment. In parts of West Africa
where SP and AQ both remain efficacious, the combination
AQ+SP could be considered for first line treatment for
uncomplicated malaria. The drugs have similar pharmacokinetic
profiles and the combination may offer a cheaper, longer lasting
and readily available alternative to ACTs, with the benefit of
longer post-treatment prophylaxis. AQ+ART is being increasingly
used and it will be interesting to see whether this combination,
with its mismatched kinetics, can prevent the development of AQ
resistance. In addition, it will be important to monitor for potential
toxicities of AQ after repeated treatment doses.
Found at: doi:10.1371/journal.pone.0001578.s001 (0.06 MB
Research Ethics Committee for approval
Found at: doi:10.1371/journal.pone.0001578.s002 (0.07 MB
Original study protocol as submitted to the Malawi
The authors would like to thank the children and their parents for taking
part in this study and the study team, Alice Mbekyani, Rose Paligolo, Mary
Kandapo, Albert Malenga, Joseph Bwanali, Andrew Khoriyo and David
Misomali for their hard work. We would also like to thank the District
Health Officer for permission to site the study at Chileka Health Centre
and all the other staff at the health centre. Thank you to Dr Sarah White
for her statistical advice and supervision of MM and to John White for
generation of the study randomisation code. We are indebted to Drs Shaf
Ahmed, Frank Bell and Grace Malenga as the study Safety Management
Committee and to Dr Neena Bodasing for acting as the study monitor.
Conceived and designed the experiments: CP EZ MM SW DB PW.
Performed the experiments: DB SN. Analyzed the data: DB MM.
Contributed reagents/materials/analysis tools: CP SW MM. Wrote the
paper: CP EZ MM SW DB PW.
SP Combination Therapies
PLoS ONE | www.plosone.org7 February 2008 | Volume 3 | Issue 2 | e1578
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SP Combination Therapies
PLoS ONE | www.plosone.org8 February 2008 | Volume 3 | Issue 2 | e1578