Assessment of the efficacy of antimalarial drugs recommended by the National Malaria Control Programme in Madagascar: up-dated baseline data from randomized and multi-site clinical trials.

Abstract

In order to improve the monitoring of the antimalarial drug resistance in Madagascar, a new national network based on eight sentinel sites was set up. In 2006/2007, a multi-site randomized clinical trial was designed to assess the therapeutic efficacy of chloroquine (CQ), sulphadoxine-pyrimethamine (SP), amodiaquine (AQ) and artesunate plus amodiaquine combination (ASAQ), the antimalarial therapies recommended by the National Malaria Control Programme (NMCP).
Children between six months and 15 years of age, with uncomplicated falciparum malaria, were enrolled. Primary endpoints were the day-14 and day-28 risks of parasitological failure, either unadjusted or adjusted by genotyping. Risks of clinical and parasitological treatment failure after adjustment by genotyping were estimated using Kaplan-Meier survival analysis. Secondary outcomes included fever clearance, parasite clearance, change in haemoglobin levels between Day 0 and the last day of follow-up, and the incidence of adverse events.
A total of 1,347 of 1,434 patients (93.9%) completed treatment and follow-up to day 28. All treatment regimens, except for the chloroquine (CQ) treatment group, resulted in clinical cure rates above 97.6% by day-14 and 96.7% by day-28 (adjusted by genotyping). Parasite and fever clearance was more rapid with artesunate plus amodiaquine, but the extent of haematological recovery on day-28 did not differ significantly between the four groups. No severe side-effects were observed during the follow-up period.
These findings (i) constitute an up-dated baseline data on the efficacy of antimalarial drugs recommended by the NMCP, (ii) show that antimalarial drug resistance remains low in Madagascar, except for CQ, compared to the bordering countries in the Indian Ocean region such as the Comoros Archipelago and (iii) support the current policy of ASAQ as the first-line treatment in uncomplicated falciparum malaria.

Full text

Page 1

BioMed Central
Page 1 of 12
(page number not for citation purposes)
Malaria Journal
Open Access
Research
Assessment of the efficacy of antimalarial drugs recommended by
the National Malaria Control Programme in Madagascar: Up-dated
baseline data from randomized and multi-site clinical trials
Didier Ménard*1, Arsène Ratsimbasoa2, Milijaona Randrianarivelojosia1,
Léon-Paul Rabarijaona2, Lucie Raharimalala1, Olivier Domarle3,
Laurence Randrianasolo2, Arthur Randriamanantena2, Martial Jahevitra1,
Valérie Andriantsoanirina1, Marie-Ange Rason1, Rogelin Raherinjafy1,
Emma Rakotomalala1, Luciano Tuseo4 and Andrianirina Raveloson5
Address: 1Malaria Unit Research, Institut Pasteur de Madagascar, Antananarivo, Madagascar, 2Epidemiology Unit, Institut Pasteur de Madagascar,
Antananarivo, Madagascar, 3Immunology Unit, Institut Pasteur de Madagascar, Antananarivo, Madagascar, 4WHO Office of Madagascar and La
Réunion, Antananarivo, Madagascar and 5National Malaria Control Programme, Ministry of Health, Antananarivo, Madagascar
Email: Didier Ménard* - dmenard@pasteur.mg; Arsène Ratsimbasoa - arsene@pasteur.mg; Milijaona Randrianarivelojosia - mili@pasteur.mg;
Léon-Paul Rabarijaona - lprabarijaona@unicef.org; Lucie Raharimalala - LRM@santenet.mg; Olivier Domarle - domarle@pasteur.mg;
Laurence Randrianasolo - laurence@pasteur.mg; Arthur Randriamanantena - arthur@pasteur.mg; Martial Jahevitra - mjahevitra@yahoo.fr;
Valérie Andriantsoanirina - valerie@pasteur.mg; Marie-Ange Rason - mieange@pasteur.mg; Rogelin Raherinjafy - raherinjafy@yahoo.fr;
Emma Rakotomalala - emma@pasteur.mg; Luciano Tuseo - tuseol@mg.who.afro.int; Andrianirina Raveloson - a_raveloson@hotmail.com
* Corresponding author
Abstract
Background: In order to improve the monitoring of the antimalarial drug resistance in
Madagascar, a new national network based on eight sentinel sites was set up. In 2006/2007, a multi-
site randomized clinical trial was designed to assess the therapeutic efficacy of chloroquine (CQ),
sulphadoxine-pyrimethamine (SP), amodiaquine (AQ) and artesunate plus amodiaquine
combination (ASAQ), the antimalarial therapies recommended by the National Malaria Control
Programme (NMCP).
Methods: Children between six months and 15 years of age, with uncomplicated falciparum
malaria, were enrolled. Primary endpoints were the day-14 and day-28 risks of parasitological
failure, either unadjusted or adjusted by genotyping. Risks of clinical and parasitological treatment
failure after adjustment by genotyping were estimated using Kaplan-Meier survival analysis.
Secondary outcomes included fever clearance, parasite clearance, change in haemoglobin levels
between Day 0 and the last day of follow-up, and the incidence of adverse events.
Results: A total of 1,347 of 1,434 patients (93.9%) completed treatment and follow-up to day 28.
All treatment regimens, except for the chloroquine (CQ) treatment group, resulted in clinical cure
rates above 97.6% by day-14 and 96.7% by day-28 (adjusted by genotyping). Parasite and fever
clearance was more rapid with artesunate plus amodiaquine, but the extent of haematological
recovery on day-28 did not differ significantly between the four groups. No severe side-effects
were observed during the follow-up period.
Published: 4 April 2008
Malaria Journal 2008, 7:55doi:10.1186/1475-2875-7-55
Received: 1 December 2007
Accepted: 4 April 2008
This article is available from: http://www.malariajournal.com/content/7/1/55
© 2008 Ménard et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Page 2

Malaria Journal 2008, 7:55http://www.malariajournal.com/content/7/1/55
Page 2 of 12
(page number not for citation purposes)
Conclusion: These findings (i) constitute an up-dated baseline data on the efficacy of antimalarial
drugs recommended by the NMCP, (ii) show that antimalarial drug resistance remains low in
Madagascar, except for CQ, compared to the bordering countries in the Indian Ocean region such
as the Comoros Archipelago and (iii) support the current policy of ASAQ as the first-line treatment
in uncomplicated falciparum malaria.
Background
Despite major efforts made by national and international
health organizations, malaria remains the most wide-
spread infectious parasitic diseases and one of the most
serious global health problems in the world [1]. In sub-
Saharan African, the main problem is Plasmodium falci-
parum drug-resistance, especially with the spread of the
parasite resistance to the inexpensive and widely used
drugs, such as chloroquine (CQ) or sulphadoxine-
pyrimethamine (SP) [2,3], and the major consequence is
the use of ineffective antimalarial drugs leading to the
increasing malarial incidence and mortality [4,5].
Therefore, substantial efforts have been made to encour-
age the monitoring and the evaluation of the antimalarial
drugs resistance in the endemic countries for assessing
regularly their antimalarial drug policies and ensuring a
continued coverage of effective antimalarial treatment [6].
In Madagascar, since 2005, antimalarial treatment is in
transition with the elaboration and the implementation
of the new national policy for the fight against malaria [7].
The main modifications in term of drugs use has been the
withdrawal of CQ in favour of artemisinin combination
therapies (ACTs), as first-line (artesunate plus amodi-
aquine combination, ASAQ) and second-line treatment
(artemether plus lumefantrine combination), and the use
of the SP for intermittent preventive treatment for preg-
nant women (IPTp). This choice was guided by the recom-
mendations of WHO and unpublished data from a
clinical trial conducted on the island of Sainte Marie in
2004, which show 36.9% of treatment failure over the 14-
day follow-up period, unadjusted by genotyping. How-
ever, with regards to the home treatment of presumed
malaria in children (HMM), it has been decided to con-
tinue to use pre-packaged chloroquine, either PaluStop®
sold by the NGO "Population Service International" (PSI)
or Ody Tazomoka® freely distributed at primary public
health facilities by Malagasy Ministry of Health (Mal-
MoH), as transitory measure until ACTs were available at
community level [8]. At present, the implementation of
the ACTs as first-line treatment at health centres level is
complete on the east coast of Madagascar, with the result
that only 24% (31/131) of the health districts in Madagas-
car are using ACT.
Since 1999, the antimalarial drug resistance surveillance
system in Madagascar is supported by a national network
for the surveillance of malaria resistance (named RER –
Réseau d'Etude de la Résistance). The RER was formed as
a collaborative effort between the MalMoH and the
Malaria Research Unit of the Institut Pasteur de Madagas-
car (IPM). The strategy of monitoring was initially based
on the use of the in vitro assessment of P. falciparum sensi-
tivity to antimalarial drugs and the evaluation of the fre-
quency of genetic markers associated with P. falciparum
drug resistance [9,10]. In vivo studies were also carried out,
but these studies were limited geographically, the sample
sizes were small and used only a 14-day follow-up period,
a series of constraints which may have significantly under-
estimated the true risk of treatment failure [11-14].
In order to improve the monitoring of antimalarial drug
resistance in Madagascar, a new RER was set up in 2006,
with the support of Global Fund. According to the WHO
recommendation [15], this network was based on sentinel
sites located within public or private health facilities and
selected to be representative of the range of ecological and
epidemiologic conditions. The main purpose of this paper
is to report the establishment of the new RER, and to
present the up-dated baseline data from multi-site rand-
omized clinical trial assessing the therapeutic efficacy of
antimalarial therapies recommended by the National
Malaria Control Programme.
Methods
Organizational structure and study sites
The antimalarial drug efficacy trials were conducted in
two steps in eight sentinel sites located in the four differ-
ent malarious epidemiological strata throughout Mada-
gascar: from February to June 2006, in Ejeda and Ihosy in
the South Madagascar (sub-desert stratum, epidemic
prone), in Maevatanana and Miandrivazo in the West
Madagascar (tropical stratum, seasonal and endemic area)
and in Tsiroanomandidy and Moramanga in the foothills
of the Central Highlands of Madagascar (highlands stra-
tum, low-endemic area); from March to August 2007 in
Andapa and Farafangana in the East Madagascar (equato-
rial stratum, perennial endemic area) (Figure 1).
Patient enrolment
This was an open-label trial in which patients with micro-
scopically-confirmed falciparum malaria were rand-

Page 3

Malaria Journal 2008, 7:55http://www.malariajournal.com/content/7/1/55
Page 3 of 12
(page number not for citation purposes)
Map of Madagascar based on malarious epidemiological strata and the eight selected sentinel sites involved for monitoring the antimalarial drug resistance [19]
Figure 1
Map of Madagascar based on malarious epidemiological strata and the eight selected sentinel sites involved for
monitoring the antimalarial drug resistance [19].










































EIR, Entomological Inoculation Rate
Ejeda
Ihosy
Farafangana
Miandrivazo
Tsiroanomandidy
Maevatanana
Moramanga
Andapa
Tropical stratum
Sub-desert stratum
Highlands stratum
Equatorial stratum
EIR = 0.2
EIR = 32
EIR = 9
EIR = 35
EIR = 9
EIR = 2
EIR = 2.5
EIR = 6
EIR = 240
EIR = 100
EIR = 0.9

Page 4

Malaria Journal 2008, 7:55http://www.malariajournal.com/content/7/1/55
Page 4 of 12
(page number not for citation purposes)
omized into three or four treatment groups according to
sentinel sites: CQ, amodiaquine (AQ), SP or ASAQ in
Ejeda, Ihosy, Miandrivazo, Maevatanana and Moram-
anga; CQ, AQ and SP in Tsiroanomandidy and AQ, SP
and ASAQ in Andapa and Farafangana.
All patients aged between six months and 15 years were
eligible to be enrolled and were screened for malaria at the
primary health centres in the sentinel sites on the basis of
history of febrile illness. According to the slightly modi-
fied WHO 2003 protocol [15], inclusion criteria were (i)
monoinfection with P. falciparum at a parasitaemia
between 1,000 and 200,000/μl, (ii) axillary temperature ≥
37.5°C, (iii) body weight > 5 kg, (iv) absence of severe
malnutrition, (v) absence of febrile conditions caused by
diseases other than malaria, (vi) absence of 'danger signs'
(inability to stand, breastfeeding or drink; recent convul-
sions; lethargy or persistent vomiting) and of severe and
complicated malaria, (vii) haemoglobin (Hb) ≥ 5 g/dl,
and (viii) informed written consent of parents/guardians.
Known hypersensitivity to SP, AQ, or AS was considered
as an exclusion criterion. Once written informed consent
was given, patients were enrolled in the study and
assigned consecutive patient numbers. Randomization to
treatment group was performed in blocks of three or four,
and treatment regimens were allocated by an independent
individual, not involved in the analysis of the study.
Treatment and follow-up procedures
Patients were administered either CQ (10 mg/kg on days
0 and 1, and 5 mg/kg on day 2), AQ (10 mg/kg on days 0,
1, and 2), SP (25 mg/kg sulphadoxine and 1.25 mg/kg
pyrimethamine as a single dose on day 0) or ASAQ (AS: 4
mg/kg on days 0, 1, and 2 and AQ:10 mg/kg on days 0, 1,
and 2). Patients were directly observed for 30 minutes
after treatment, and the dose was readministered if vomit-
ing occurred. Patients who repeatedly vomited their first
dose of study medication were excluded from the study.
Patients were assessed on days 1, 2, 3, 7, 14, 21 and 28,
and any intervening day they were unwell for malaria
infection. Blood was obtained by finger prick on all fol-
low-up days and on any unscheduled day to use for anal-
ysis of thick and thin blood smears and for storage on
filter paper. Thick and thin blood slides were examined by
light microscopy for parasites on any day during the 28-
day follow-up. Blood slides were read by a microscopist
blind to treatment allocation. All slides were controlled by
a second microscopist also blind to treatment group and
previous diagnosis. Discordant slides were read, blind to
treatment group and previous diagnosis, by a third micro-
scopist. Haemoglobin was measured on Day 0 and Day 28
using a HemoCue haemoglobinometre (HemoCue AB,
Ängelholm, Sweden).
Outcome measures
Treatment outcomes were assessed according to WHO
2003 guidelines as Early Treatment Failure (ETF; danger
signs or complicated malaria or failure to adequately
respond to therapy on days 0–3), Late Clinical Failure
(LCF; danger signs or complicated malaria or fever and
parasitemia on days 4–28 without previously meeting cri-
teria for ETF), Late Parasitological Failure (LPF; asympto-
matic parasitaemia on days 4–28 without previously
meeting criteria for ETF or LCF), and Adequate Clinical
and Parasitological Response (ACPR; absence of parasi-
taemia on day 28 without previously meeting criteria for
ETF, LCF, or LPF) [15]. Overall Treatment failure (OFT)
was considered as the sum of the ETP, LCT and LPF.
Patients classified as having suffered treatment failure
were treated with quinine (10 mg/kg three times daily for
seven days); however, their response to repeat therapy was
not assessed. Patients were excluded after enrolment if any
of the following occurred: (i) use of antimalarial drugs
outside of the study protocol; (ii) detection during follow-
up of mixed malarial infections (iii) parasitaemia in the
presence of a concomitant febrile illness which would
interfere with the classification of treatment outcome; (iv)
withdrawal of consent; (v) loss to follow-up, (vi) protocol
violation, or (vii) death due to a non-malaria illness.
Laboratory procedures
Blood smears were stained with 10 % Giemsa for 10 min.
Parasite densities were determined from thick blood
smears by counting the number of asexual parasites per
200 WBCs (or per 500, if the count was less than 10 para-
sites/200 WBCs), assuming a WBC count of 8,000/μl. A
smear was considered negative if no parasites were seen
after review of 100 fields. Thin blood smears were used to
detect non-falciparum infections [16].
Molecular genotyping techniques were used to distin-
guish recrudescence from new infection for all patients
failing therapy after day 7. Filter paper blood samples col-
lected on the day of enrolment, on day 1 and on the day
of failure were analysed for polymorphisms in the genes
for merozoite surface protein-1 (msp-1) and merozoite
surface protein-2 (msp-2) using nested-PCR as previously
described [17]. First, msp-2 genotyping patterns on the day
of failure were compared with those at treatment initia-
tion and on day 1, using Quantity One© software (BioRad
laboratories, Inc., 1000 Alfred Nobel Drive, Hercules, CA
94547, United States). If all of the msp-2 alleles present on
the day of failure were present at the time of treatment ini-
tiation or on day 1, genotyping was repeated using msp-1.
An outcome was defined as recrudescence if all msp-1 and
msp-2 alleles present at the time of failure were present at
the time of treatment initiation or on day 1, and defined
as a new infection otherwise.

Page 5

Malaria Journal 2008, 7:55http://www.malariajournal.com/content/7/1/55
Page 5 of 12
(page number not for citation purposes)
Statistical analysis
Data were entered and verified using EpiInfo 6.04© soft-
ware (Centers for Disease Control and Prevention,
Atlanta, Georgia, United States), and analysed using Med-
Calc© software version 9.1.0.1 (MedCalc Software, Broek-
straat 52, 9030 Mariakerke, Belgium). As the proportion
of treatment failures was unknown in the sentinel sites, a
minimum of 50 patients was included for each treatment
group and each site, according to available human and
financial resources and the WHO recommendations [15].
Efficacy data were assessed with a per-protocol analysis
that included all patients who completed the study. An
age-stratified analysis for patients less than five years of
age and patients five years or older was planned. This
study was not designed for the primary analysis to be strat-
ified by site. Parasite densities were normalized using log-
arithmic transformation. Categorical variables were
compared using χ2 or Fisher's exact test, and continuous
variables were compared using an independent samples t
test.
The primary efficacy outcomes were 14-day and 28-day
clinical and parasitological failure risks both unadjusted
and adjusted by genotyping. Risks of clinical and parasito-
logical treatment failure after adjustment by genotyping
were estimated using Kaplan-Meier survival analysis tech-
niques in accordance with the new WHO protocol [15].
With survival analysis, data were censored for new infec-
tions. Secondary outcomes included fever clearance, para-
site clearance, change in haemoglobin levels between Day
0 and the last day of follow-up, and the incidence of
adverse events. Hypothesis testing was made using risk
differences, exact 95% confidence intervals, and P values.
A P value (two-tailed) of less than 0.05 was considered
statistically significant.
Ethical approval
The study protocol was reviewed and approved by the Eth-
ics Committee of the Ministry of Health of Madagascar
(N°007/SANPF/2007). An informed written consent was
provided by the parents/guardians of all patients before
they were included in the study.
Results
Enrolment
In the eight sentinel sites, 8,363 patients were screened,
with 1,873 patients positive for P. falciparum (22.4%). Of
these patients, a total of 1,434 patients were enrolled in
February-June 2006 and March-July 2007 (76.6%). A total
of 320 patients were randomized to CQ, 385 to AQ, 383
to SP, and 346 to ASAQ. The main reasons for patients
with P. falciparum not being included were non-consent,
inability to attend follow-up or concurrent disease. The
flow of patients through the trial is shown in Figure 2.
A total of 1,347 of 1,434 patients (93.9%) completed
treatment and follow-up to day 28 (92.5% in the AQ
group, 93.7% in the CQ group, 94.2% in the SP group,
and 95.4% in the ASAQ group). Eighty eight patients were
either lost to follow-up (n = 68) or excluded/withdrawn
from the trial (n = 20). Of these patients, 18 withdrew
consent, two were withdrawn due to adverse events. The
baseline characteristics of patients across the treatment
groups were similar at each site (Table 1).
Primary outcome: treatment efficacy
The results of the treatment efficacy are presented by treat-
ment group and day of follow-up in Table 2 and by age
group and study site in Tables 3 and 4. Figure 3 showed
the Kaplan-Meier curve of cumulative treatment failure
over the 28-day follow-up period, adjusted by genotyping.
All treatment regimens, except for the CQ treatment
group, resulted in clinical cure rates above 97.6% by day-
14 and 96.7% by day-28 (adjusted by genotyping). Con-
sidering all sites, the OTF of the CQ group was signifi-
cantly higher than in the other treatment group as well as
day 14 or day 28 unadjusted or adjusted by genotyping (P
< 0.0001).
In the CQ group, ETF was uncommon with only 10.6% of
the OTF (i.e., 14/132). Most of the treatment failures
adjusted by genotyping were occurred in the second/third
week of follow-up mainly as LPF (63.6%). Among the
eight sites, the OTF adjusted by genotyping of CQ was sig-
nificantly different ranging from 19% in Ihosy to 64% in
Ejeda (P < 0.0001). The risk of treatment failure adjusted
by genotyping in children less than five years old was
almost twice as frequent than in older patients (52.7% vs.
37.3%, adjusted odds ratio [OR], 1.8; 95% confidence
interval [CI], 1.2–3.0; P = 0.004). The risk of treatment
failure by day-14 was significantly lower than by day-28
adjusted by genotyping (32.0% vs. 44.0%, risk difference
12.0%, 95% CI: 4.3%–19.5%, P = 0.003).
In the SP group, treatment failures were rare with an OTF
adjusted by genotyping of 3.3%, but ETF was accounting
for more than one third of the treatment failures: Two
patients from Ejeda developed danger signs despite
decreasing parasite density, one patient from Tsiroano-
mandidy presented at day 3, a parasite density > 25% of
count on day 0 and the last patient from Maevatanana, a
parasite density with an axillary temperature ≥ 37.5°C.
The distribution of the treatment failure adjusted by gen-
otyping was not significantly different among the sites (P
= 0.08), especially because of the small number of the
patients included per site and the small number of treat-
ment failure.

Page 6

Malaria Journal 2008, 7:55http://www.malariajournal.com/content/7/1/55
Page 6 of 12
(page number not for citation purposes)
Flow of patients
Figure 2
Flow of patients.

8363 Patients
screened
1873 Patients
with microscopically confirmed
Plasmodium falciparum malaria
1434 Randomized
202 in Ejeda
273 in Miandrivazo 222 in Maevatanana
139 in Tsiroanomandidy 161 in Moramanga
102 in Andapa 87 in Farafangana
248 in Ihosy
439 Excluded
95 Refused consent
344 Did not meet Inclusion criteria
320
Randomized to
Receive Chloroquine
385
Randomized to
receive Amodiaquine
383
Randomized to receive
Sulfadoxine/Pyrimethamine
346
Randomized to receive
Artesunate/Amodiaquine
Status at follow-up Day 14
309 Evaluable
10 Lost to follow-up
1 Withdrawn
Status at follow-up Day 14
372 Evaluable
10 Lost to follow-up
3 Withdrawn
Status at follow-up Day 14
368 Evaluable
12 Lost to follow-up
3 Withdrawn
Status at follow-up Day 14
335 Evaluable
11 Lost to follow-up
0 Withdrawn
Status at follow-up Day 28
300 Evaluable
6 Lost to follow-up
3 Withdrawn
Status at follow-up Day 28
361 Evaluable
1 Lost to follow-up
6 Withdrawn
Status at follow-up Day 28
330 Evaluable
5 Lost to follow-up
0 Withdrawn
300 Included in primary
analysis At Day 28
50 in Ejeda
58 in Ihosy
66 in Miandrivazo
52 in Maevatanana
38 in Tsiroanomandidy
36 in Moramanga
0 in Andapa
0 in Farafangana
356 Included in primary
analysis At Day 28
48 in Ejeda
53 in Ihosy
66 in Miandrivazo
53 in Maevatanana
39 in Tsiroanomandidy
36 in Moramanga
32 in Andapa
29 in Farafangana
361 Included in primary
analysis At Day 28
50 in Ejeda
59 in Ihosy
64 in Miandrivazo
51 in Maevatanana
40 in Tsiroanomandidy
38 in Moramanga
29 in Andapa
30 in Farafangana
330 Included in primary
analysis At Day 28
50 in Ejeda
57 in Ihosy
65 in Miandrivazo
50 in Maevatanana
0 in Tsiroanomandidy
38 in Moramanga
41 in Andapa
29 in Farafangana
Status at follow-up Day 28
356 Evaluable
13 Lost to follow-up
4 Withdrawn

Page 7

Malaria Journal 2008, 7:55http://www.malariajournal.com/content/7/1/55
Page 7 of 12
(page number not for citation purposes)
In the AQ and ASAQ groups, treatment efficacies were glo-
bally very high throughout Madagascar. Treatment fail-
ures adjusted by genotyping were not significantly
different among the sentinel sites (P = 0.24 for AQ and P
= 0.12 for ASAQ).
Table 1: Baseline characteristics by treatment group*
Treatment Group
Characteristics Chloroquine AmodiaquineSulphadoxine-PyrimethamineArtesunate-Amodiaquine
No. of patients enrolled
Ejeda
Ihosy
Miandrivazo
Maevatanana
Tsiroanomandidy
Moramanga
Andapa
Farafangana
Age
median (range), y
Proportion of children ≤ 5 years
Weight, median (IQR), kg
Female
Temperature, mean (95% CI)
Trophozoite density, geometric mean (range),
parasite/μl
Haemoglobin, median (IQR), g/dL
Anemic at enrollment±
320
51
62
67
55
43
42
0
0
385
51
62
69
56
48
39
32
28
383
50
61
69
56
48
40
29
30
346
50
63
68
55
0
40
41
29
5 (0.5 – 15)
142 (45.4)
15 (6 – 58)
158 (49.5)
38.4 (38.3 – 38.5)
15,302
(1,241 – 200,000)
10.1 (5.3 – 15.8)
107 (39.3)
5 (0.5 – 15)
185 (48.7)
15 (5 – 62)
188 (48.8)
38.4 (38.3 – 38.5)
133,068
(1,000 – 194,000)
10.2 (5.8 – 15.6)
114 (34.5)
5 (0.5 – 15)
182 (47.8)
14 (6 – 60)
186 (48.6)
38.4 (38.3 – 38.5)
13,880
(1,000 – 196,000)
10.0 (5.6 – 14.7)
127 (38.5)
5 (0.5 – 15)
167 (48.7)
13 (2.6 – 56)
169 (49.0)
38.6 (38.5 – 38.7)
14,346
(1669 – 192000)
10.1 (5.1 – 14.5)
103 (35.4)
Abbreviations: CI, confidence interval; IQR, interquartile range.
*Data are presented as No. (%) unless otherwise indicated.
†Anaemia defined as haemoglobin of less than 10 g/dL.
Table 2: Treatment outcomes stratified by treatment group and day of follow-up
Outcome Measure by DayNo./Total (%) of Patients [95% CI]
CQSP AQASAQ
Day 14
Clinical failureETF
LCF
LPF
14/309
29/309
56/309
99/309
(4.5)
(9.4)
(18.1)
(32.0)
[2.6–7.3]
[6.5–13.0]
[14.1–22.7]
[27.0–37.4]
4/368
2/368
3/368
9/368
(1.1)
(0.5)
(0.8)
(2.4)
[0.3–2.6]
[0.1–1.7]
[0.2–2.2]
[1.2–4.4]
2/372
0/372
0/372
2/372
(0.5)
(0)
(0)
(0.5)
[0.1–1.7] 0/335
0/335
0/335
0/335
(0)
(0)
(0)
(0)
Parasitological failure
Overall Treatment
failure
[0.1–1.7]
Day 28 Unadjusted by genotyping
Clinical failure ETF
LCF
LPF
14/300
45/300
104/300
163/300
(4.7)
(15.0)
(34.7)
(54.4)
[2.7–7.5]
[11.3–19.4]
[29.4–40.2]
[48.7–59.9]
4/361
4/361
8/361
16/361
(1.1)
(1.1)
(2.2)
(4.4)
[0.4–2.7]
[0.4–2.7]
[1.0–4.2]
[2.6–6.9]
2/356
3/356
6/356
11/356
(0.6)
(0.8)
(1.7)
(3.1)
[0.1–1.8]
[0.2–2.3]
[0.7–3.5]
[1.6–5.3]
0/330
4/330
8/330
12/330
(0)
(1.2)
(2.4)
(3.6)
[0.4–2.9]
[1.1–4.6]
[2.0–6.1]
Parasitological failure
Overall Treatment
failure
Day 28 Adjusted by genotyping
Clinical failureETF
LCF
LPF
14/300
34/300
84/300
132/300
(4.7)
(11.3)
(28.0)
(44.0)
[2.7–7.5]
[8.1–15.3]
[23.1–33.3]
[38.5–49.7]
4/361
3/361
5/361
12/361
(1.1)
(0.8)
(1.4)
(3.3)
[0.4–2.7]
[0.2–2.2]
[0.5–3.0]
[1.8–5.6]
2/356
1/356
3/356
6/356
(0.6)
(0.3)
(0.8)
(1.7)
[0.1–1.8]
[0–1.4]
[0.2–2.3]
[0.7–3.5]
0/330
2/330
4/330
6/330
0
(0.6)
(1.2)
(1.8)
[0.4–2.7]
[0.1–2.0]
[0.4–2.9]
[0.7–3.7]
Parasitological failure
Overall Treatment
failure
CI: Confidence Interval; ETF: Early Treatment Failure; LCF: Late Clinical Failure; LPF: Late Parasitological Failure; CQ: Chloroquine; AQ:
Amodiaquine; ASAQ: Artesunate + Amodiaquine; SP: Sulphadoxine-Pyrimethamine

Page 8

Malaria Journal 2008, 7:55http://www.malariajournal.com/content/7/1/55
Page 8 of 12
(page number not for citation purposes)
Secondary outcomes
Parasite clearance was more rapid with ASAQ than CQ,
AQ or SP until day 3 (P < 0.0001). Fever clearance was
delayed with CQ and SP, the proportion of febrile patients
being significantly lower with ASAQ and AQ until day-2.
The details are shown on Figure 4. On day-28, the extent
of haematological recovery (median of individual
increases in Hb) did not differ significantly between the
four groups (CQ, 0.8 g/dl, -4.1 to 4.9; SP, 1.0 g/dl, -4.8 to
6.4; AQ, 1.0 g/dl, -4.4 to 8.3; ASAQ, 0.9 g/dl, -6.0 to 6.4).
No severe side-effects attributable to the study medication
were observed during the follow-up period. Minor side
effects as vomiting were reported between day 1 and day
3 in seventeen patients (1.26%): 8 in the AQ group, 4 in
the CQ and the SP groups and 1 in the ASAQ group.
Discussion
As effective case management remains the cornerstone of
malaria elimination [18], the monitoring of antimalarial
drugs resistance is essential to ensure a continued cover-
age of effective antimalarial treatment. With the support
of resources from the Global Fund, the main objective of
the new RER was to set up an expanded programme to
monitor antimalarial drug resistance in Madagascar,
based on sentinel sites. The selected sentinel sites were
chosen at peripheral level (within public or private health
facilities) to be representative of the four epidemiological
strata in which the country could divided [19]. Because of
the size of the country and the availability of financial and
human resources, two sites were selected by epidemiolog-
ical stratum for establishing a surveillance system for in
vivo drug efficacy. Seven additional sites were secondly
incorporated in the RER for monitoring in vitro drug resist-
ance (in vitro drug sensitivity assay and molecular mark-
ers) to provide complementary data and warning signals
of the possible emergence of resistance or trends of declin-
ing drug efficacy.
The objective of the new RER was firstly to establish up-
dated baseline data on the efficacy of the antimalarial
drugs recommended by the MalMoH in its new Madagas-
car national policy. The methodology used was based on
the WHO 2003 protocol [15], with some modifications
(compromise between the high transmission and the low
to moderate transmission protocols): (i) children
enrolled in the study were aged between six months and
15 years and (ii) of P. falciparum parasitaemia on inclu-
sion was between 1,000 and 200,000/μl. The protocol
included randomized, 28-day follow-up with genotyping
to discriminate recrudescence from new infections, and
assessment of safety and tolerability. Despite the complex-
ity of conducting a multi-site study in Madagascar, an
Table 3: Treatment failures by Day 28 (adjusted by genotyping), stratified by age group
Treatment groups No. of treatment failure/Total of Patients (%) [95% CI]
Age groupsSignificance level P*
0.5 – 4 years
≥ 5 years
CQ
SP
AQ
ASAQ
69/131 (52.6) [44.1–61.1]
8/171 (4.7) [2.2–8.7]
4/174 (2.3) [0.7–5.5]
4/161 (2.8) [0.8–5.9]
63/169 (37.3) [30.2–44.7]
3/190 (1.6) [0.4–4.2]
2/182 (1.1) [0.2–3.6]
2/169 (1.2) [0.2–3.9]
0.01
0.57
0.64
0.51
CQ: Chloroquine; AQ: Amodiaquine; ASAQ: Artesunate + Amodiaquine; SP: Sulphadoxine-Pyrimethamine.
CI: Confidence Interval
Chi-square test for the comparison of two proportions
Table 4: CQ Treatment failures by Day 28 (adjusted by genotyping), stratified by age group and study site
CQNo. of treatment failure/Total of Patients (%) [95% CI]
0.5 – 4 years
≥ 5 years All age
TropicalMaevatanana
Miandrivazo
Moramanga
Tsiroanomandidy
Ejeda
Ihosy
19/28 (67.9) [49.1–83.0]
10/16 (62.5) [37.6–83.2]
10/26 (38.5) [21.5–57.9]
13/21 (61.9) [40.2–80.5]
14/20 (70.0) [47.7–86.8]
3/20 (15.0) [4.0–35.6]
9/24 (37.5) [20.1–57.8]
16/50 (32.0) [20.2–45.8]
3/10 (30.0) [8.3–62.0]
9/17 (52.9) [29.7–75.2]
18/30 (60.0) [41.9–76.2]
8/38 (21.1) [10.3–36.1]
28/52 (53.8) [40.3–67.0]
26/66 (39.4) [28.2–51.5]
13/36 (36.1) [21.8–52.6]
22/38 (57.9) [41.9–72.7]
32/50 (64.0) [50.1–76.4]
11/58 (19.0) [10.4–30.6]
Highlands
Sub-desert
CQ: Chloroquine. CI: Confidence Interval. Chi-square test for the comparison of two proportions

Page 9

Malaria Journal 2008, 7:55http://www.malariajournal.com/content/7/1/55
Page 9 of 12
(page number not for citation purposes)
excellent completion rates was achieved (> 93%) and
high-quality data were collected.
The four main antimalarial drugs used and recommended
by the NMCP were evaluated in five sites. In Tsiroano-
mandidy, ASAQ was not evaluated because this drug was
already assessed in another clinical trial [20] and in
Andapa and Farafangana, CQ was not evaluated for ethi-
cal reasons because of the too high frequency of treatment
failure found in the previous clinical trial in 2006.
Two methodological points could be underline by the
results of the study: (1) the necessity to extend the follow-
up to a minimum of 28 days (even more for drugs with a
prolonged duration of action) to avoid the underestima-
tion of the risk of treatment failure [21], i.e. for CQ, most
of the treatment failures occurred between 15 and 28 days
after treatment, a 14 day follow-up will underestimated
the OTF of 12% (32.0% for 14-days instead of 44.0% for
28-days) and (2) the necessity to enrol sufficient number
of patients to allow the stratification of the result based on
age (< 5 years and ≥ 5 years), i.e. the risk of CQ treatment
failure in children < 5 years was 2-fold more frequent than
in older patients (five to 15 years old), especially in mod-
erate to high transmission areas (Maevatanana and Mian-
drivazo).
This multi-site study represents the first extent in vivo drug
efficacy study performed in Madagascar since 1983 [22-
24]. The CQ efficacy was signifantly different between the
eight sites and ranged from moderate (Ihosy, 81%), inter-
mediate (Miandrivazo, 60.6% and Moramanga, 63.9%)
to low (Maevatanana, 46.2%; Tsiroanomandidy, 42.1%
and Ejeda, 36%), showing the important of the multi-site
studies. However, the results about the CQ efficacy were
comparable to those found previously in the last in vivo
trial carried out in Sainte Marie on the east coast in 2004
(36.9% of clinical failure within two weeks of CQ treat-
ment). Currently, CQ remains the drug most widely avail-
able (distribution and financial criteria) and is the first
drug used in most of areas in Madagascar at community
level, for treating acute fever before or without laboratory
diagnosis. According to Hastings et al [6], even with 50%
of efficacy, CQ might still be perceived as very effective at
community level, especially because of its antipyretic
activity that alleviate symptoms and because that most of
the recrudescence's which occur two to three week later
after the treatment are not perceived as a failure. Based on
these findings, the MalMoH must improve the HMM, as
Kaplan-Meier curve of cumulative treatment failure over the 28-day follow-up period, adjusted by genotyping
Figure 3
Kaplan-Meier curve of cumulative treatment failure over the 28-day follow-up period, adjusted by genotyping.
0%
0
5%
10%
10
15%
20%
20
25%
30%
30
35%
40%
40
45%
50%
50
0
0
7
7
14
14
21
21
28
28
AQ
ASAQ
CQ
SP
CQ
SP
AQ
ASAQ
Day of follow-up
Cumulative incidence of treatment failure, %
No. at risk
CQ
SP
AQ
ASAQ
320
383
385
346
274
365
373
236
205
354
357
332
162
350
352
328
137
346
344
318

Page 10

Malaria Journal 2008, 7:55http://www.malariajournal.com/content/7/1/55
Page 10 of 12
(page number not for citation purposes)
soon as possible, by withdrawing pre-packaged CQ and
gradually replacing it with pre-packaged ASAQ.
The results concerning the SP efficacy were more inform-
ative, contrasting with the good effectiveness reported in
previous studies in 2003–2004 [13,14]. The more worry-
ing was to finding even if the treatment failures were rare
that the ETFs were accounting for more than one third of
the SP treatment failure. There is no doubt that with the
increased drug use of SP in the next few years in Madagas-
car as IPT for pregnant women, the monitoring of its drug
resistance especially with the useful and reliable molecu-
lar makers such as Pf-dhfr and Pf-dhps [25] will be make it
a priority for the RER.
This study also confirmed that ASAQ, the first-line treat-
ment of the uncomplicated malaria recommended in
Madagascar was very effective, particularly because of the
excellent effectiveness of AQ, the partner drug associated
with artesunate. Although it was not feasible to monitor
for the potentially serious adverse effects associated with
amodiaquine such as neutropenia or hepatotoxicity (the
sample size was too small to exclude any clinically toxicity
with a low frequency such as < 1:100), ASAQ was well tol-
erated in the study population. This combination pro-
Proportions of patients with asexual parasites and fever (T° ≥ 37.5°C) on days of follow-up by treatment group
Figure 4
Proportions of patients with asexual parasites and fever (T° ≥ 37.5°C) on days of follow-up by treatment group.
Asexual parasites
0
20
40
60
80
100
01237
Day of follow-up
Percentage of patients
AQ
ASAQ
CQ
SP
Fever
0
20
40
60
80
100
01237
Day of follow-up
Percentage of patients
AQ
ASAQ
CQ
SP

Page 11

Malaria Journal 2008, 7:55http://www.malariajournal.com/content/7/1/55
Page 11 of 12
(page number not for citation purposes)
duced also faster parasite and fever clearances than the
other antimalarial treatments. These findings constitute
important baseline data on the efficacy of this combina-
tion, a key tool within the framework of the elimination
of the malaria [18].
The results from this multi-site study show that antimalar-
ial drug resistance remains low in Madagascar, except for
CQ, compared to the bordering countries in the Indian
Ocean region such as the Comoros Archipelago [26,27].
The next step must be to complete these data by providing
the frequency of mutant parasites correlated with the CQ-
resistance (Pfcrt) or SP-resistance (Pf-dhfr or Pf-dhps) or
the in vitro response of parasites for drug for which no val-
idate molecular marker are available such as amodi-
aquine, artemisinin derivatives or amino alcohols
(quinine, lumefantrine). These complementary indicators
should allow the early detection of the spread of P. falci-
parum drug resistance, especially those introduced from
the Comoros Islands [28]. Indeed, the extension of the
RER at regional level is also crucial to enable the countries
of the region to share national information on antimalar-
ial drug efficacy and to help to minimize delays in imple-
menting national antimalarial drug policy change.
Authors' contributions
DM contributed to the design and coordination of the
study, supervised the enrolment and follow up of the
patients, assisted with data entry and interpretation and
prepared the manuscript. ARat supervised the enrolment
and follow up of the patients. LR, ARan and RR performed
field work in Tsiroanomandidy and Moramanga. M-AR
and ER were involved in laboratory work. MJ and VA car-
ried out molecular genotyping. MR, L-PR, LR, OD, LT and
ARav helped to compose the manuscript and gave con-
structive advice.
Acknowledgements
The authors thank all the patients and their parents or guardians for partic-
ipating in the study. We would also like to thank the clinical study teams: in
Ejeda (Dr Diamondra Raveloariseheno, Mme Brunette Razanadrazanina, M.
Leo Paul Andriamahazo), in Ihosy (Dr Vony Rabekotonirina, Mlle Mino
Andrianjafy, M. Jean Bruno Velo Jasmin), in Farafangana (Dr Didier Ignace
Ralaizandriny, Dr Jean Yvon Niel Mahadimby, Mme Noeline Rasoarilalao) In
Miandriavazo (Dr Didier Ignace Ralaizandriny, M. Mamisoa Judicaël Rako-
tonaivo, M. Jacquinot Mangolo), in Moramanga (Dr Arthur Randriamanan-
tena, Mme Noeline Rasoarilalao, M. Rianala Nohary Harimanana
Andrianina, M. Andriarajonantenaina Zakaherizo Ramiandrasoa), in
Tsiroanomandidy (Dr Arsène Ratsimbasoa, Dr Laurence Randrianasolo, M.
Rogelin Raherinjafy), in Maevatanana (Dr Hanitra Ranaivoson, M. Jacquet
Randriamboavonjy, M. Patrick Rabemanantsoa) and in Andapa (Dr Andri-
arajonantenaina Zakaherizo Ramiandrasoa, M. Perlinot Herindrainy, Mme
Brunette Razanadrazanina). We would also like to thank all the health
workers at the health centres of Ejeda (Dr Andriamandiby Petterson),
Ihosy (Sœur Joany Ravaoary), Farafangana (Dr Ralalaharisoa Laurence),
Miandrivazo (Dr Aurelien Ravelojoeliandriambeloaritafika), Moramanga
(Dr Marie Claudine Ranaivoarisoa), Tsiroanomandidy (Dr Monique Rajeri-
son and Sœur Emmanuella Randriatavy), Maevatanana (Dr Tsimahohy
Zanah Rahasivelo) and Andapa (Dr Hector) and their respective district
administrations, for allowing us to conduct these studies and working
alongside the study teams for lengthy periods.
Financial support: This work was supported by the Global Fund to Fight
AIDS, Tuberculosis and Malaria round 3 grant MDG-304-G05-M and the
IAEA project, RAF-6-025-9002.
References
1.Snow RW, Guerra CA, Noor AM, Myint HY, Hay SI: The global dis-
tribution of clinical episodes of Plasmodium falciparum
malaria. Nature 2005, 434(7030):214-217.
2.Attaran A, Barnes KI, Curtis C, d'Alessandro U, Fanello CI, Galinski
MR, Kokwaro G, Looareesuwan S, Makanga M, Mutabingwa TK, Tal-
isuna A, Trape JF, Watkins WM: WHO, the Global Fund, and
medical malpractice in malaria treatment. Lancet 2004,
363(9404):237-240.
3.White NJ: Antimalarial drug resistance. J Clin Invest 2004,
113(8):1084-1092.
4.Korenromp EL, Williams BG, Gouws E, Dye C, Snow RW: Measure-
ment of trends in childhood malaria mortality in Africa: an
assessment of progress toward targets based on verbal
autopsy. Lancet Infect Dis 2003, 3(6):349-358.
5.Trape JF: The public health impact of chloroquine resistance
in Africa. Am J Trop Med Hyg 2001, 64(1-2 Suppl):12-17.
6.Hastings IM, Korenromp EL, Bloland PB: The anatomy of a
malaria disaster: drug policy choice and mortality in African
children. Lancet Infect Dis 2007, 7(11):739-748.
7.Ministère de la Santé et du Planning Familial: Politique National de
Lutte contre le Paludisme. 2005:p 44.
8.Ratsimbasoa A, Randrianarivelojosia M, Millet P, Soares JL, Rabari-
jaona L, Rakotoson B, Malvy D, Menard D: Use of pre-packaged
chloroquine for the home management of presumed
malaria in Malagasy children. Malar J 2006, 5:79.
9.Ariey F, Randrianarivelojosia M, Duchemin JB, Rakotondramarina D,
Ouledi A, Robert V, Jambou R, Jahevitra M, Andrianantenaina H,
Raharimalala L, Mauclere P: Mapping of a Plasmodium falci-
parum pfcrt K76T mutation: a useful strategy for controlling
chloroquine resistance in Madagascar. J Infect Dis 2002,
185(5):710-712.
10.Randrianarivelojosia M, Rakotonjanabelo LA, Mauclere P, Ratsim-
basoa A, Raharimalala LA, Ariey F: National Network study to
perpetuate the surveillance of Plasmodium falciparum sensi-
tivity to antimalarials in Madagascar. Arch Inst Pasteur Madagas-
car 2002, 68(1-2):73-78.
11.Raharimalala LA, Rabarijaona L, Randrianarivelojosia M, Razanavololo
F, Rason MA, Andrianantenaina HB, Andrianaivolambo L, Rakotoni-
aina JC, Leong Pock Tsi JM, Rajaonarivelo E, Leon T, Duchemin JB,
Ariey F: Malaria study in the cyclone risk zone: entomological
, diagnostic and therapeutic approach in the southeastern
region of Madagascar. Arch Inst Pasteur Madagascar 2002, 68(1-
2):79-85.
12.Randriamanantena A, Randrianasolo L, Vonimpaisomihanta JA,
Tafangy PB, Bayant Z, Randrianarivelojosia M: Therapeutic efficacy
of amodiaquine against uncomplicated malaria in Madagas-
car. Sante 2007, 17(2):75-78.
13.Randrianasolo L, Randriamanantena A, Ranarivelo L, Ratsimbasoa A,
Domarle O, Randrianarivelojosia M: Monitoring susceptibility to
sulfadoxine-pyrimethamine among cases of uncomplicated,
Plasmodium falciparum malaria in Saharevo, Madagascar.
Ann Trop Med Parasitol 2004, 98(6):551-554.
14.Randrianasolo L, Randriamanantena A, Ratsimbasoa A, Rakotoson JD,
Randriambelosoa J, Raveloson A, Rakotondrajaona N, Tuseo L, Ran-
drianarivelojosia M: Assessment of sulfadoxine-pyrimethamine
(Fansidar, Paludar) efficacy in patients with uncomplicated
malaria in Madagascar: preliminary study to propose a sim-
plified study protocol. Arch Inst Pasteur Madagascar 2003, 69(1-
2):52-56.
15.World Health Organization: Assessment and monitoring of anti-
malarial drug efficacy for the treatment of uncomplicated
falciparum malaria. Geneva: WHO 2003.
16.World Health Organization: Management of Uncomplicated
Malaria and the Use of Antimalarial Drugs for the Protection

Page 12

Publish with BioMed Central and every
scientist can read your work free of charge
"BioMed Central will be the most significant development for
disseminating the results of biomedical research in our lifetime."
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central
yours — you keep the copyright
Submit your manuscript here:
http://www.biomedcentral.com/info/publishing_adv.asp
BioMedcentral
Malaria Journal 2008, 7:55http://www.malariajournal.com/content/7/1/55
Page 12 of 12
(page number not for citation purposes)
of Travellers. Report of an Informal Consultation (WHO/
MAL/96). Geneva, Switzerland, WHO 1996.
Cattamanchi A, Kyabayinze D, Hubbard A, Rosenthal P, Dorsey G:
Distinguishing recrudescence from reinfection in a longitudi-
nal antimalarial drug-efficacy study: Comparison of results
based on genotyping of msp-1, msp-2, and glurp. Am J Trop
Med Hyg 2003, 68:133-139.
Ministère de la Santé du Planning Familial et de la Protection Sociale :
Plan Stratégique de Lutte Contre Le Paludisme. Du contrôle
vers l’élimination du paludisme à Madagascar : Extension et
consolidation des zones indemnes du paludisme (2007-
2012). 2007:p 54.
Mouchet J, Blanchy S: Particularities and stratification of
malaria in Madagascar. Sante 1995, 5(6):386-388.
Randrianarivelojosia M, Randrianasolo L, Ratsimbasoa A, Albert Same
Ekobo S, Ama Moor V, Kuété T, Brasseur P, Agnamey P, Ndiaye JL,
Faye B, Gaye OS I., Traoré A, Dicko Y: A multinational, ran-
domised comparative study to assess the tolerability and
efficacy of Coarsucam® (fixed dose combination of artesu-
nate plus amodiaquine) once or twice daily versus Coar-
tem® (fixed dose combination of artemether plus
lumefantrine) for uncomplicated Plasmodium falciparum
malaria. : Amsterdam, The Netherlands. ; 2007.
Stepniewska K, Taylor WR, Mayxay M, Price R, Smithuis F, Guthmann
JP, Barnes K, Myint HY, Adjuik M, Olliaro P, Pukrittayakamee S,
Looareesuwan S, Hien TT, Farrar J, Nosten F, Day NP, White NJ: In
vivo assessment of drug efficacy against Plasmodium falci-
parum malaria: duration of follow-up. Antimicrob Agents Chem-
other 2004, 48(11):4271-4280.
Deloron P, Ramanamirija JA, Le Bras J, Coulanges P: 4-Aminoquin-
oline-sensitivity of Plasmodium falciparum in Madagascar.
III. Studies in three regions of the interior. Arch Inst Pasteur
Madagascar 1984, 51(1):57-68.
Deloron P, Ramanamirija JA, Le Bras J, Verdier P, Coulanges P: 4-
Aminoquinoline-sensitivity of Plasmodium falciparum in
Madagascar. I. Study in two regions of the west coast. Arch
Inst Pasteur Madagascar 1984, 51(1):29-42.
Deloron P, Ramanamirija JA, Verdier F, Coulanges P: 4-Aminoquin-
oline-sensitivity of Plasmodium falciparum in Madagascar. II.
Study in two regions of the east coast. Arch Inst Pasteur Mada-
gascar 1984, 51(1):43-55.
Kublin JG, Dzinjalamala FK, Kamwendo DD, Malkin EM, Cortese JF,
Martino LM, Mukadam RA, Rogerson SJ, Lescano AG, Molyneux ME,
Winstanley PA, Chimpeni P, Taylor TE, Plowe CV: Molecular mark-
ers for failure of sulfadoxine-pyrimethamine and chlorpro-
guanil-dapsone treatment of Plasmodium falciparum
malaria. J Infect Dis 2002, 185(3):380-388.
Parola P, Pradines B, Simon F, Carlotti MP, Minodier P, Ranjeva MP,
Badiaga S, Bertaux L, Delmont J, Morillon M, Silai R, Brouqui P, Parzy
D: Antimalarial drug susceptibility and point mutations asso-
ciated with drug resistance in 248 Plasmodium falciparum
isolates imported from Comoros to Marseille, France in
2004 2006. Am J Trop Med Hyg 2007, 77(3):431-437.
Silai R, Moussa M, Abdalli Mari M, Astafieva-Djaza M, Hafidhou M,
Oumadi A, Randrianarivelojosia M, Said Ankili A, Said Ahmed B, Gay-
ibor AH, Ariey F, Ringwald P: Surveillance of falciparum malaria
susceptibility to antimalarial drugs and policy change in the
Comoros. Bull Soc Pathol Exot 2007, 100(1):6-9.
Menard D, Randrianarivo-Solofoniaina AE, Said Ahmed B, Jahevitra M,
Andriantsoanirina V, Ranjalahy Rasolofomanana J, Rabarijaona LP:
Drug-Resistant Malaria Parasites Introduced into Madagas-
car from Comoros Islands. Emerg Infect Dis 2007,
13(11):1759-62.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.