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A Randomized Open-Label Trial of Artesunate-
Sulfadoxine-Pyrimethamine with or without Primaquine
for Elimination of Sub-Microscopic
P. falciparum
Parasitaemia and Gametocyte Carriage in Eastern Sudan
Badria El-Sayed
1
*, Salah-Eldin El-Zaki
1
, Hamza Babiker
2
, Nahla Gadalla
1
, Tellal Ageep
1
, Fathi Mansour
1
, Omer Baraka
3
, Paul Milligan
4
, Ahmed
Babiker
1
1 Department of Epidemiology, Tropical Medicine Research Institute, National Centre for Research, Khartoum, Sudan, 2 Faculty of Medicine, Sultan
Qaboos University, Muscat, Oman, 3 Faculty of Medicine, University of Khartoum, Khartoum, Sudan, 4 Department of Epidemiology and Population
Health, London School of Hygiene and Tropical Medicine, London, United Kingdom
Background. In areas of seasonal malaria transmission, treatment of asymptomatic carriers of malaria parasites, whose
parasitaemia persists at low densities throughout the dry season, could be a useful strategy for malaria control. We carried out a
randomized trial to compare two drug regimens for clearance of parasitaemia in order to identify the optimum regimen for use in
mass drug administration in the dry season. Methodology and Principal Findings. A two-arm open-label randomized controlled
trial was conducted during the dry season in an area of distinct seasonal malaria in two villages in Gedarif State in eastern Sudan.
Participants were asymptomatic adults and children aged over 6 months, with low-density P. falciparum infection detected by PCR.
Participants were randomized to receive artesunate/sulfadoxine-pyrimethamine (AS+SP) combination for three days with or
without a dose of primaquine (PQ) on the fourth day. Parasitaemia detected by PCR on days 3, 7 and 14 after the start of treatment
and gametocytes detected by RT-PCR on days 7 and 14 were then recorded. 104 individuals who had low density parasitaemia at
screening were randomized and treated during the dry season. On day 7, 8.3% were positive by PCR in the AS+SP+PQ group and
6.5% in the AS+SP group (risk difference 1.8%, 95%CI 210.3% to +13.8%). At enrolment, 12% (12/100) were carrying gametocytes.
This was reduced to 6.4% and 4.4% by day 14 (Risk difference 1.9% (95%CI 29.3% to +13.2%) in AS+SP+PQ and AS+SP groups,
respectively. Conclusion. Addition of primaquine to artemisinin combination treatment did not improve elimination of
parasitaemia and prevention of gametocyte carriage in carriers with low-density parasitaemia in the dry season. Trial
Registration. ClinicalTrials.gov NCT00330902
Citation: El-Sayed B, El-Zaki S-E, Babiker H, Gadalla N, Ageep T, et al (2007) A Randomized Open-Label Trial of Artesunate- Sulfadoxine-Pyrimethamine
with or without Primaquine for Elimination of Sub-Microscopic P. falciparum Parasitaemia and Gametocyte Carriage in Eastern Sudan. PLoS ONE 2(12):
e1311. doi:10.1371/journal.pone.0001311
INTRODUCTION
In malaria endemic countries of sub-Saharan Africa, the majority
of Plasmodium falciparum infections are asymptomatic and remain
untreated. In eastern Sudan people who become infected during
the wet season may retain chronic sub-microscopic asymptomatic
infections throughout the dry season [1–3]. The presence of
gametocytes within these sub-microscopic infections has been
demonstrated using sensitive gametocyte-specific RT-PCR [4–6].
In a cohort study of 38 individuals monitored monthly by RT-
PCR, 40% were found to retain gametocytes throughout the dry
season [7]. Treatment of carriers to clear parasitaemia during the
dry season may reduce the source of infection available to
mosquitoes that emerge at the start of the rainy season. This could
contribute to malaria control strategy if high coverage with an
effective therapy is achieved.
Artemisinin combination treatment is highly effective in
eliminating asexual parasitaemia, the source of merozoites
committed for gametocyte production [8]. Since gametocyte
longevity is limited [9] such treatment may lead, in the absence of
reinfection, to eventual elimination of gametocyte carriage
[10,11]. Three doses of artesunate were found to reduce
gametocyte carriage after treatment in clinical malaria cases
[12]. Artemisinin derivatives inhibit gametocyte development but
are not effective against mature gametocytes. Therefore to achieve
more rapid clearance of mosquito-infective stages from the blood
stream an actively gametocytocidal drug may be required [13].
Primaquine has been used for half a century as a hypnozoitocidal
drug against Plasmodium vivax, as a prophylactic against all malaria
species, and as a gametocytocidal drug against P. falciparum
[14,15]. The World Health Organization has recommended, for
some areas, addition of a single dose of primaquine to treatment
regimens for P. falciparum malaria to reduce transmission [16].
Individuals with low gametocyte densities undetectable by
microscopy may still be infectious to mosquitoes [17,18]. The
Academic Editor: Piero Olliaro, World Health Organization, Switzerland
Received October 10, 2007; Accepted October 10, 2007; Published December 12,
2007
Copyright: ß 2007 El-Sayed 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 received technical and financial support from the joint WHO
Eastern Mediterranean Regional Office (EMRO), Division of Communicable
Diseases (DCD), and the WHO Special Programme for Research and Training in
Tropical Diseases (TDR): The EMRO/TDR Small Grants Scheme for Operational
Research in Tropical and other Communicable Diseases (grant no. SGS 04/64, to B.
B. El Sayed), TC grant SUD 06/21 from the International Atomic Energy Agency
(IAEA) and the National Centre for Research and Ministry of Science and
Technology, Sudan to TMRI. The sponsors did not contribute to the design of the
study or interpretation of the data.
Competing Interests: The authors have declared that no competing interests
exist.
* To whom correspondence should be addressed. E-mail: badriab2@yahoo.com
PLoS ONE | www.plosone.org 1 December 2007 | Issue 12 | e1311
combination of PCR and RT-PCR techniques allows the
detection of very low density gametocyte producing P. falciparum
infections [4], which persist as asymptomatic infections during the
dry season. The present study was undertaken to compare the
efficacy of AS+SP and AS+SP+PQ in treating low-density sub-
microscopic P. falciparum parasitaemia before the transmission
season in an area of marked seasonal transmission in eastern
Sudan in order to identify the optimum regimen for use in mass
drug administration during the dry season.
METHODS
The protocol for this trial and supporting CONSORT checklist
are available as supporting information; see Checklist S1 and
Protocol S1.
Participants
The study was carried out in two villages in eastern Sudan.
Abunaja Juama located at about 18 km south west of Gedarif and
Kanara at 7 km south of Gedarif.
The whole area is characterized by seasonal malaria with
transmission confined to three months of the year, October –
December. The main malaria parasite is P. falciparum and the main
mosquito vector is Anopheles arabiensis [2,19,20]. The proposal,
informed consent form and the project information sheet were
reviewed and approved by the Ethical Committee of the Federal
Ministry of Health, Sudan. Preliminary meetings were held with
the community leaders to ask for their permission. All adults and
children aged 6 months or above who were resident in the two
villages were invited to participate and to be screened for the
presence of asymptomatic P. falciparum. Signed informed consent
forms were obtained from all participants and from the parents or
guardians of children under 15 years. Exclusion criteria included
pregnancy, history of allergy to sulpha drugs, fever or other signs or
symptoms of illness but none of the persons screened had any of
these conditions. After screening individuals were excluded if, on
microscopic examination of a blood film, Plasmodium species other
than falciparum were detected or the sample was negative by PCR.
Interventions
Participants were randomized to receive a standard dose of AS
and SP (AS+SP) over three days, or AS+SP over the first three
days plus primaquine (PQ) administered on the fourth day.
Objectives
We wanted to determine the efficacy of AS+SP compared to
AS+SP+PQ in clearing sub-patent parasitaemia and gametocyte
carriage in persons with asymptomatic sub-microscopic P.
falciparum parasitaemia, in order to identify the optimum regimen
for use in mass drug administration in the dry season.
Outcomes
The outcomes were P.falciparum parasitaemia detected by PCR on
days 3, 7 and 14 and presence of gametocytes detected by RT-
PCR on days 7 and 14. For these endpoints, a sample by finger
prick was obtained on days 0, 3, 7 and 14 on glass slides and on
filter paper, and on days 7 and 14 venous blood was collected in
K
3
EDTA vacutainers. Adverse events were recorded on days 1, 2,
3, 7 and 14 and packed Cell Volume was measured on days 0, 7
and 14. Parasite DNA was extracted by the Chelex-Resin method
[21]. A nested PCR method was employed to detect the small sub-
unit ribosomal RNA gene (ssrRNA) [22] as a sensitive screening
method for sub-patent parasitaemia. For detection of gametocytes
by reverse transcriptase PCR (RT-PCR), mRNA of P. falciparum
gametocyte specific pfS25 gene was amplified in a nested PCR to
increase sensitivity of the detection [4]. Thin and thick blood
smears were prepared from all samples and stained in Giemsa’s
stain for ten minutes. Films were examined by two experienced
technicians. Slides were considered negative if no parasite stage
was detected after completing 100 negative high power fields.
Sample size
In a previous trial [23] 30% of subjects were gametocyte positive on
day 7 after treatment with SP and 3 doses of artesunate. Assuming a
similar rate, a trial with 60 subjects in each arm would have at least
90% power (using a significance level of 0.05) to detect a difference
between treatments if gametocyte prevalence detected by RT-PCR
after treatment is 30% or more on day 14 in the AS+SP arm and 5%
or less in AS+SP+PQ, allowing for 10% loss to follow up.
Randomization—Sequence generation and
allocation concealment
A cross sectional survey was carried out in the middle of the dry
season (June) 2004 to identify sub-patent parasite carriers. A finger
prick sample was collected on a glass slide for microscopic
diagnosis and on filter paper for molecular analysis. Persons who
were positive by PCR in June and eligible were enrolled in the
study and randomly allocated into one of the two treatment arms.
The list of carriers was sorted according to village and age to
ensure that the treatment groups were balanced with respect to
these two variables. The random allocation of this ordered list into
the treatment arms was then created using restricted randomiza-
tion with a block size of 12 in Stata version 7 (Statacorp, Texas). In
August, a clinical examination was performed; no participants
were excluded due to illness or other exclusion criteria. Before
administering the first treatment dose (day 0), a venous blood
sample was obtained from each individual in K
3
EDTA vacutainer
tubes (Becton Dickinson) for gametocyte detection by RT-PCR,
and a drop of blood was collected on a glass slide for microscopic
diagnosis and another drop on filter paper for PCR.
Randomization—Implementation
Treatment was administered under medical supervision. SP was
administered on day 0 as 25 mg/kg sulfadoxine/1.25 mg/kg
pyrimethamine for children under 50 kg, while adults and children
weighing 50 kg or more were given three tablets (each tablet
contained 500 mg of sulfadoxine and 25 mg of pyrimethamine).
Artesunate was given on days 0, 1 and 2 at a dose of 200 mg (two
tablets at 100 mg) for adults and for children weighing 50 kg or
more, while children under 50 kg received 4 mg/kg body weight.
Participants randomized to receive primaquine were, in addition,
given a single dose of 0.75-mg/kg body weight on day 3. After each
treatment dose, patients were observed for one hour, if the drug was
vomited during the first half hour a full dose was repeated, if
vomiting occurred after one hour half a dose was repeated.
Blinding
The trial was not blinded; however laboratory staff performing the
PCR assays were unaware of the treatment allocation.
Statistical methods
95% confidence intervals were calculated for the difference
between the proportions with parasitaemia and gametocytaemia
in the two groups on days 7 and 14, using the modified score
method of Newcombe ([24], method 10). Packed cell volume was
P. falciparum Parasitaemia
PLoS ONE | www.plosone.org 2 December 2007 | Issue 12 | e1311
compared using analysis of covariance, adjusting for the baseline
measurement. Stata version 9 (Statacorp, Texas) was used.
RESULTS
Participant flow
Out of a total of 615 asymptomatic individuals screened by
microscopy in June 2004 (the dry season) 12 were positive for
malaria parasites (9 were P. falciparum, 2 were P. vivax and one was
P. ovale). 114/615 (19%) had PCR detectable P. falciparum
parasitaemia (including the 9 which were positive by microscopy)
and were therefore eligible for enrolment into the trial. The three
who were positive by microscopy for other Plasmodium species were
PCR negative for falciparum.
The trial was started in August (the pre-transmission season) when
104 eligible participants were enrolled and 10 were absent. Of these
4 (4.2%) were positive for asymptomatic P. falciparum by microscopy.
Five people withdrew consent; 2 in the AS+SP withdrew by day 3.
While for the AS+SP+PQ group one withdrew on day 0 after the first
dose and two by day 7 as shown in the trial profile Fig 1.
Recruitment
Participants were enrolled from 17
th
to 20
th
August, and followed
up until 3
rd
September 2004.
Baseline data
The demographic data for the study group is shown in Table 1.
Numbers analyzed
Four (4%) individuals from those who were randomized in the
AS+SP group were found to be negative by PCR on day 0. Of
these 3 were lost to follow up on day 14 and one withdrew consent
by day 3. They have been excluded from the analysis of safety and
efficacy. The remaining withdrawn and lost to follow-up cases
were also excluded. Data analysis was performed according to the
protocol.
Outcomes and estimation
On recruitment (day 0) 100 (96%) out of 104 individuals carried
asymptomatic sub-patent P. falciparum infection. However, only 20
(20.4%) out of 98 participants seen on day 3 post-treatment were
found to harbour P. falciparum infection detectable by PCR. On
day 7 only seven (7.4%) out of 94 participants retained PCR
detectable P. falciparum infection, 4/48 (8.3%) in the AS+SP+PQ
group and 3/46 (6.5%) in the AS+SP group (risk difference 1.8%,
95%CI 210.3% to +13.8%). Similarly, five out of 92 seen on day
14 had PCR detectable parasitaemia (Table 2).
With regard to gametocytes, on enrolment, twelve (12%) out of
100 individuals carried gametocytes detectable by RT-PCR. None
of them were gametocyte positive on day 7 or day 14. The same
Figure 1. CONSORT Flowchart
doi:10.1371/journal.pone.0001311.g001
Table 1. Characteristics of participants at enrolment.
......................................................................
AS+SP (N = 52) AS+SP+PQ (N = 52)
Age in years (mean, range) 18.9 (4–76) 24.8 (4–84)
Male: Female 22:30 23:29
P.falciparum detected by PCR 43 (83%) 52 (100%)
Gametocytes detected by RT-PCR 5/43 (10%) 6/52 (11.5%)
P. Falciparum detected by microscopy 0/52 (0%) 4/52 (8%)
doi:10.1371/journal.pone.0001311.t001
...................................
P. falciparum Parasitaemia
PLoS ONE | www.plosone.org 3 December 2007 | Issue 12 | e1311
persons who were detected positive by PCR on days 7 and 14 were
also positive for gametocytes by RT-PCR. On day 7, four (8.3%)
out of 48 on AS+SP+PQ and three (6.5%) out of 46 individuals in
AS+SP were found to have RT-PCR detected gametocytes (risk
difference 1.9%, 95% CI: 28.5% to 12.3%). Similarly on day 14,
6.4% in AS+SP+PQ and 4.4% in AS+SP had sub-microscopy
gametocytaemia (risk difference 1.9% (95%CI 29.3% to +13.2%).
Two out of the seven gametocyte carriers detected on day 7
retained their gametocyte producing infection until day 14.
However, gametocytes reappeared on day 14 among three
individuals who were gametocyte negative on day 7.
Packed cell volume was similar in both groups on day 7, mean
34.6% (15–44%) in the AS+SP group and 34.2% (26–42%) in the
AS+SP+PQ group, difference between groups adjusted for
baseline 0.78 (20.75,2.3) P = 0.315. Similar results were seen on
day 14 (Table 3).
Adverse events
No serious or severe adverse events were reported. Four of the
participants (3.8%) vomited after the first treatment dose of
AS+SP; 1 on AS+SP and 3 on AS+ SP+PQ arm. One of these
vomited immediately, refused to take the drug again and withdrew
consent. The other three cases (2.9%) vomited after more than
eight hours after taking the drug and the dose was therefore, not
repeated for them. Two people complained of insomnia and
another two complained of itching.
DISCUSSION
Interpretation
We compared the efficacy of AS+SP and AS+SP+PQ in clearing
asymptomatic sub-patent P. falciparum parasitaemia that persist in
the dry season, as a potential control strategy in areas of seasonal
transmission. After treatment with AS+SP alone 80% (78/98) of
subjects with asymptomatic sub-patent P. falciparum infections
became PCR negative by day 3 (before administration of PQ).
However, only three (6.4%) in AS+SP and four (8.3%) in
AS+SP+PQ group had sub-patent gametocytes on day 7, and
two (4.4%) and three (6.4%) on day 14, respectively.
The present results are consistent with previous findings that
asymptomatic sub-patent parasitaemia [1–3] and gametocytes
carriage [6,7] persist throughout the dry season among inhabitants
of villages in eastern Sudan. The low prevalence of gametocytes
compared to asexual stages has been reported previously. The ratio
of gametocyte to asexual stages in P. falciparum was found to be less
than 1:10 [25–27], a recent study calculates a much lower ratio
(1:156) [28]. The positive correlation found between gametocyte
density in the blood and infectiousness to mosquitoes [29–32] is
considered to be controversial. It may be hampered by the low
sensitivity of microscopy [33] and by transmission blocking
immunity [34]. P. falciparum gametocyte carriers in nature usually
harbour less than 100 gametocyte ml
21
blood and there is evidence
that 1–10 gametocytes ml
21
blood are infectious to mosquitoes [17].
Therefore, the dry season sub-patent carriers represent an infectious
source of P. falciparum to Anopheles mosquitoes [35] upon their
reappearance during the rainy season. This has recently been
demonstrated by a mosquito infectivity study in Kenya, which
revealed high contribution of inhabitants with sub-patent P.
falciparum infection to infectivity of Anopheles mosquitoes [35].
Our results show the efficacy of artesunate against asymptom-
atic sub-patent gametocyte carriage. Gametocytes present before
treatment were most probably mature since we used the PfS25
gene, which is exclusively expressed by mature gametocytes
[36,37]. Gametocyte sequestration could be the main reason for
detection of some RT-PCR positive samples on days 7 and 14 [9].
It has been suggested that SP can release gametocytes from sites of
sequestration, a process that can increase gametocyte density on
day 7 to 14 post-treatment [38], but this was not borne out by
results from a randomized trial in asymptomatic carriers [11].
However these gametocytes are not expected to be a major source
for malaria transmission as, in absence of asexual forms, may live
for an average of about 6.4 days [27]. This assumption is
supported by the fact that five out of seven participants who were
gametocyte positive on day 7 became negative by day 14. In spite
of the safety of primaquine reported in this study, the low
frequency of gametocytes did not allow us to demonstrate its
importance conclusively but our results suggest that AS+SP
without primaquine is an adequate regimen to clear the pre-
transmission season gametocyte reservoir, which plays a central
role in secondary cases arising at the start of the transmission
season. Control strategies which target gametocyte carriage during
the dry season could have strong impact on malaria morbidity and
mortality in this area.
Generalizability
The clearance of sub-patent parasitaemia and gametocyte carriage
by day 7 after treatment with AS+SP indicates that this may be an
Table 2. Sub-patent parasitaemia and gametocyte carriage before and after Treatment.
..................................................................................................................................................
Group Day 0 Day 3 Day 7* Day 14
AS+SP Parasitaemia detected by PCR 84.6% (44/52) 25.5% (12/47) 6.5% (3/46) 4.4% (2/45)
Gametocytaemia detected by RT-PCR 11.5% (6/52) - 6.5% (3/46) 4.4% (2/45)
AS+SP+PQ Parasitaemia detected by PCR 100% (52/52) 15.7% (8/51) 8.3% (4/48) 6.4% (3/47)
Gametocytaemia detected by RT-PCR 11.5% (6/52) - 8.3% (4/48) 6.4% (3/47)
*
Risk difference between the two treatment groups on day 7: 1.9% (95%CI 28.5% to +12.3%), on day 14: 2% (95%CI 27.2% to +11.2%).
doi:10.1371/journal.pone.0001311.t002
..................................
Table 3. Mean Packed Cell Volume in the two treatment
groups.
......................................................................
Day of follow up
SP+AS %
(N)
SP+AS+PQ %
(N) Difference*
Day 0 35.7 (42) 36.1 (47)
Day 7 34.7 (39) 34.2 (40) 0.78 (20.75,+2.3) P = 0.32
Day 14 35.4 (34) 35.2 (41) 0.86 (20.31,+2.0) P = 0.15
*
Differences were estimated from regression, with the day 0 included as a
covariant.
doi:10.1371/journal.pone.0001311.t003
......................................
P. falciparum Parasitaemia
PLoS ONE | www.plosone.org 4 December 2007 | Issue 12 | e1311
effective treatment for use to clear low density P.falciparum
parasitaemia in the dry season without the need for the addition
of primaquine. These results, for infections near the limit of
detection, may not apply to higher density infections where
detectable gametocytes may persist for several weeks in spite of
effective treatment of asexual parasitemia [38], although gameto-
cytes will disappear over time when the asexual population has
been eliminated. A limitation of the study is the sensitivity of the
PCR that was used. PCR negative individuals may test positive if
more sensitive methods are used and may still be able to infect
mosquitoes. Another limitation is that the power to detect an effect
of PQ on gametocyte carriage was limited.
Overall evidence
The combination AS+SP without primaquine is effective in
eliminating the dry season sub-microscopic parasitaemia and
gametocyte carriage. Therefore, this regimen could be recom-
mended for use in mass drug administration in the dry season to
control malaria in areas of seasonal transmission.
However, it may be necessary to complete the drug adminis-
tration before the rainy season and resurgence of the mosquitoes
so as to avoid transmission of mature gametocytes.
SUPPORTING INFORMATION
Protocol S1 Trial Protocol
Found at: doi:10.1371/journal.pone.0001311.s001 (0.16 MB
DOC)
Checklist S1 CONSORT Checklist
Found at: doi:10.1371/journal.pone.0001311.s002 (0.04 MB
DOC)
ACKNOWLEDGMENTS
We acknowledge the contribution of field and laboratory staff of Tropical
Medicine Research Institute (TMRI), especially Dr. Abdel Muhsin A.
Abdel Muhsin for his help with PCR technique. We thank the residents of
Abunaja Juama and Kanara villages who participated in the study. We also
thank the National Malaria Control Programme for the drugs.
Author Contributions
Conceived and designed the experiments: PM BE OB AB. Performed the
experiments: SE NG TA FM. Analyzed the data: PM BE. Wrote the
paper: PM HB BE NG OB AB. Other: Established RT-PCR technique in
Sudan: HB. Enroled patients and was responsible for collection of patient
data during screening, enrolment and follow up, molecular analysis of
samples and data entry: SE. Contributed to patient enrolment and follow
up: FM TA.
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