Therapeutic Efficacy of Artesunate-Amodiaquine and
Polymorphism of Plasmodium Falciparumk13-Propeller
Gene in Pala (Tchad)
Issa Mahamat Souleymane1,2*, Kerah Hinzoumbé Clément1, Mbaitoloum Modobé Denis1, Ako Aristide Berenger
2, Coulibaly Baba2, Toure André Offianan2, Djimadoum Mbanga3, Tchonfiene Passiri4, Djimrassengar Honoré5, Yameogo V. Jean Marie5, Bouzid Samir6, Ringwald Pascal7, Dosso Mireille8 and Djaman Allico Joseph9
1Chad National Malaria Control Programm (NMCP)
2Department of Paludology-Mycology, Institute Pastor of Côte d’Ivoire
3University of N’Djaména, Faculty of Medicine, NCBT
4Emergency service of Pala Hospital
5WHO / Tchad
6PALAT / UNDP
7GMP / WHO, Geneva, Switzerland
8University of Félix Houphouët-Boigny, Abidjan - Institute Pastor of Côte d’Ivoire
9University of Félix Houphouët-Boigny, Abidjan, Department of Biochemistry- Institute Pastor of Côte d’Ivoire
Issa Mahamat Souleymane,Programme National de Lutte contre le Paludisme au Tchad. mail:
Received: August 29, 2017; Accepted: October 16, 2017; Published: October 30, 2017
Citation: Souleymane ISSA M, Clément KH, Denis MM, et al. (2017) Therapeutic Efficacy of Artesunate-Amodiaquine and Polymorphism of Plasmodium Falciparumk13-Propeller Gene in Pala (Tchad). Int J Open Access Clin Trials 1(1) : 1-6.
ACTs was recommended as a first-line treatment for uncomplicated
Plasmodium falciparum malaria in many malaria-endemic countries.
Regular monitoring of ACTs is recommended by the World Health
Organization (WHO) to help early detection of resistant parasites
strains and contain their rapid spread. The aim of this study was to
assess therapeutic efficacy of Artesunate-Amodiaquine (ASAQ) the
first line treatment of uncomplicated falciparummalaria in Chad and
analyze the polymorphism of Kelch13-propeller gene.
A single-arm prospective study of a 28-days follow-up was
conducted among children aged 6- 59 months with uncomplicated P.
falciparum malaria at Pala site from November to December 2015.The
primary outcome was ACPR PCR-corrected at day28 and the secondary
endpoints were the Parasite Clearance (PCT), Fever Clearance Time
(FCT) and tolerability of the drug Kelch13-propeller was amplified
and sequenced in all Plasmodium falciparum isolates.
A total of 58 children were enrolled and 51 reached the study
endpoint. Crude Adequate and clinical response was 98% at day 28
and after correction PCR this rate was 100%. Treatment was well
tolerated. No mutations neither synonymous nor non synonymous
were detected on k13 gene, after alignment with the reference
ASAQ was proved to be efficacious and well tolerated in Pala
children and no mutation was observed in the Kelch 13-propeller
gene. Further studies are needed across the country to enhance
Key Word: Malaria, P. falciparum, Artesunate-Amodiaquine, k13-
Malaria remains one of the most important public health
challenges in the world. Malaria is the leading cause of morbidity
and mortality in Africa, particularly in children under 5 years
old and pregnant women. Artemisinin-Based Combination
Treatments (ACTs) are now the recommended first-line
treatments for uncomplicated falciparum malaria worldwide.
The worldwide use of ACTs has contributed in recent years to
a substantial reduction in deaths related to falciparum malaria.
Resistance to artemisinin however has emerged in Southeast Asia
Monitoring the efficacy of ACTs becomes particularly
important in the light of emergence of artemisinin resistance
in South-East Asia [6,5].Artemether-Lumefantrine (AL) and
Artesunate-Amodiaquine (ASAQ) are widely available drugs,
which are recommended by most malaria endemic countries in
the treatment of uncomplicated falciparum malaria . Chad
National Malaria Control Programm (NMCP) recommends,
since 2005, ASAQ and AL respectively as first and second line
treatments for uncomplicated P. falciparum malaria. The World
Health Organization recommends a regular assessment of the
efficacy of the first- and second-line antimalarial drugs for an
early detection and prevention of the spread of resistant parasite
In vivo therapeutic efficacy study is the gold standard for
detecting the emergence and spread of malaria drug resistance.
Discovery of mutations in the Kelch-13 propeller region protein in
correlation with delayed clearance phenotype is a major advance.
Surveillance of artemisinin resistance to date relied on in vivo
studies to measure early clearance of peripheral parasitaemia
by light microscopy and K13 propeller gene mutations. Since the
introduction of ACTs in 2004 in Chad, very few studies have been
conducted on ACTs efficacy and Kelch13-propeller gene [9,20]. The
aim of this study was to assess the in vivo therapeutic efficacy of
Artesunate-Amodiaquine (ASAQ) and analyze the polymorphism
of the Kelch 13 propeller gene conferring artemisinin-resistance
to Plasmodium falciparumat the Pala hospital.
This study was a prospective, one-arm assessment of clinical
and tolerability of ASAQ according to WHO guidelines . Follow
up was for 28 days. The study was conducted at Pala site country
in the Mayo-Kebbi west region (9° 21’00 “N; 14 ° 58’00” W) of
Chad from November to December2015.
In the study site malaria transmission occurs from July to
December during the raining season. The majority of malaria
cases in the area is caused by P. falciparum, while Anopheles
gambiaes.s. and to a lesser extent Anopheles funestus are the
major vectors. The key malaria control interventions in the
district include use of LLNIs, malaria case management with ACTs
Intermittent Preventive Treatment during pregnancy (IPTp).
To assess the treatment effect on parasites mutations that
modulate treatment response, mutation in k13 propeller gene
the molecular marker associated with decrease artemisinin
sensitivity were investigated [10,11]. The resistance was
investigated by examining polymorphisms in the k13 propeller
domain at day 0. The method used was nested PCR protocol
followed by Sanger Sequencing using primers specific to
P.falciparum. The amplicon used for sequencing covered 740 pb
which included the k13 propeller domain .
Children aged from 6 to 59 months presenting to the facility
were enrolled if mono-specific P. falciparum infestation was
confirmed by microscopy with parasite density between 1000
and 200000 asexual parasites / μL of blood and they had fever
axillary ≥ 37.5 ° C, or history of fever over the last 24 hours. The
others inclusion criteria were ability to take oral medications;
able to come to come to health facility for follow -up; informed
consent of parent or legal guardian. Children with severe malaria
symptoms according to the WHO case definition and symptoms of
severe malnutrition and chronic diseases or with mixed infection
were excluded .
Treatment was three-day oral regimen dosed by weight
according to the manufacturer’s instructions: ASAQ Winthrop® 5
to < 9 kg: one tablet/day of Artesunate (AS) 25 mg/Amodiaquine
(AQ) 67.5 mg; 9 to < 18 kg: one tablet/day of AS 50 mg/AQ 135
mg; 18 to < 36 kg: 1 tablet/ day of AS 100 mg/AQ 270 mg.
Children who vomited during the observation period were
retreated with the same dose of medicine and observed for an
additional 30 minutes. Children with repeated vomiting were
excluded and were treated according National Control Program
treatment guidelines and excluded from the study. All children
were allowed use of antipyretics.
Children enrolled in the trial was followed up for 28 days.
Children was seen after the day of enrollement (day 0) on days
1, 2, 3, 7, 14, 21 and 28. At each day visit children were clinically
examined by a study physician who recorded findings in a Case
Report Form (CRF). Parasitaemia (asexual and sexual) was
assessed on days 1, 2, 3, 7, 14, 21, 28 and any day within the
28 days follow-up period that the child is brought to the health
facility with fever.
Thick and thin blood smears were stained with 5% Giemsa for
30 minutes. Parasitaemia was determined by reading the thick
blood smear and counting the number of asexual parasites per
200 White Blood Cells (WBCs), assuming a WBC count of 8000/
ul. Slides were considered negative if no parasite was found after
reading 100 high-powered fields. Presence of gametocytes was
All blood samples were read by two qualified independent
microscopist. Slides were quality controlled at the Swiss
Tropical Institute and Public Health. Discordance was defined as
differences between the first and second microscopist regarding
parasite density >50%, species diagnosis or any difference that
affected recruitment or study outcome. The first or second
reading was taken as final depending on whichever agrees with
the third reading.
Filter paper blots were collected at day 0 and at recurrence of
parasitaemia for PCR genotyping.
Merozoite surface proteins 1 and 2 (msp1&msp 2) and
Glutamate-Rich Protein (glurp) were used to distinguish reinfection
At each follow-up visit, any new or worsening symptom was
assessed. An adverse event was defined as any unfavorable and
unintended sign, symptom or disease temporally associated
with the use on investigational product, not present at day 0, but
occurred during follow –up, or was present at day 0 but became
worse during follow-up. Serious adverse event was defined as
any event that resulted in patient hospitalization, death, lifethreatening
experience, persistent /significant disability or
specific medical surgical intervention to prevent serious outcome.
Treatments outcomes were classified based on clinically
and parasitological outcomes assessment as recommended by
WHO .Therapeutic responses on day 28 were classified as
either Adequate Clinical And Parasitological Response (ACPR),
or Treatment Failure (TF) designated as Early Treatment Failure
(ETF), Late Clinical Failure (LCF), or Late Parasitological Failure
(LPF).The primary outcome endpoint was ACPR, corrected for re
infection using PCR genotyping at day 28.
DNA extraction and PCR
The parasite DNA was extracted from the blood sampled on
filter paper on D0 and at failure by a Qiagen DNA Mini Kit (Qiagen,
Valencia.CA) according to the manufacturer’s instructions.
The k13 gene and msp2 were amplified by a Polymerase Chain
Reaction (PCR). ForPfk13 gene, amplified PCR products were
send to GENEWIZ in UK for Sanger sequencing. Subsequent
analysis of delivered sequences was executed in comparison with
the PF3D7_1343700 sequence. The following co don positions
were checked for mutations [10,11].
Data management and analysis were completed with Epi Info
6.0.4 adaptedc to the Who excel-based applications .BioEdit
was used for sequences analysis and GraphPad Prism 5 (one way
ANOVA test) was used to compare the 3 mean temperatures from
Day 0 to Day 2.
Profile of Study Patients
During the study period from November to December2015,
163 patients were examined for uncompleted malaria at the
Urban Health Center in Pala. Among them, 58 were randomized
and 105 were excluded from the study for the reasons detailed on
the study profile (Figure1).
Figure 1: Study profile
Characteristics of the study
Of the 58 randomized patients, 32 (30.5%) were female and
26 (24.8%) were male, with a sex ratio of 0.81; Meanage (ds) 2.9
(1.3%), average weight (ds) 13.2 (3.6%), average temperature
(ds) 38 (0.9%) and mean parasite density of 3.840 asexual
parasites/μL on the day of enrolment (Table1).
Table 1: Baselines characteristics of the study
Included n (%)
58 (35.6 %)
Male n (%)
26 (44.8 %)
Female n (%)
32 (55.2 %)
59 months n (%)
8 (13.8 %)
< 59 months n (%)
50 (86.2 %)
Mean age (± SD) years
2.9 ( ±1.3)
Age (min - max) years
0.7 - 5
Mean weight (± SD) kg
13.2 (± 3.6)
Weight (min - max) kg
5 - 21
Mean axillary temperature (±SD)° C
38 (± 0.9)
Axillary temperature (min - max)° C
36.2 - 40.3
Mean parasitemia (asexual parasites / μL)
Parasitemia (min - max) asexual parasites / μL
1.040 - 20.000
A total of 58 patients were included and 51 patients were
analyzed per protocol. Prior to PCR correction, 50 patients were
ACPR that is 98. % treatment success with only one (2.0 %) Late
Clinical Failure. After PCR correction, the failure reported on Day
28 was in fact a re-infestation. Thus, all treated patients were 100
% ACPR on Days 28 (Table 2).
Table 2: Therapeutic response at D28
Patients screenedat D28
Late clinical failure
Adequate Clinical and Parasitological Response at D28
Adequate Clinical and Parasitological Response at D28 after PCR
Fever and parasites clearance time
Fever decreased very significantly during treatment period,
from day 0 to day 2. On the day of enrollment, axillary temperature
run from a minimum of 36.2° C (history of fever) to a maximum
of 38° C (Table1).
Tolerability and safety
A predominance of loss of appetite (10.3%) followed by cough
(7%) abdominal pain and vomiting (3.5%) were reported (Figure
2).No deaths and no cases of severe malaria were seen during the
study. All adverse events observed were mild (Figure 2).
Figure 1: Adverse events
Polymorphism of the K13-propeller gene
The K13-propeller gene was amplified and sequenced in
58 isolates of P. falciparum. After alignment with the reference
sequence PF3D7_1343700, all were of the wild type, without
Malaria remains a major public health problem in developing
countries. Prompt access to effective antimalarial treatment such
as Artemisinin Based-Combination Therapies (ACT) proves to be
an essential tool for controlling the disease. In this study, ASAQ was
proved to be effective treatment for uncomplicated falciparum
malaria, as evidenced by a PCR-corrected parasitological efficacy
of 100 %.The present study was conducted to provide supporting
evidence for the clinical efficacy of ASAQ, which was adopted
and implemented with AL as anti-malarial drug policy in Chad
since 2005. The efficacy assessment of ACT has also shown a
high efficacy level of ASAQ in neighboring countries of Chad.
In Nigeria, a bordering country of Chad, Oguche et al, found a
polymerase chain reaction-corrected parasitologic cure rates on
Day 28 of 98.3% with ASAQ . In the same country a ACPRcorrected
results on day 28 was 95.8% has been observed with
ASAQ during a stuy conducted by Falade et al, . In Central
African Republic, another bordering country, a 28-day therapeutic
efficacy study of ASAQ conducted by Djalle et al, in Bangui
indicated 93% of ACPR-corrected at day 28 . It seemed that
ASAQ is more efficacious in Chad than in Central African Republic
potentially due to drug pressure higher in this country. Some
studies conducted elsewhere in Africa showed good efficacy
of ASAQ [11,15,16,17].In addition to high cure rates, rapid PCT
and FCT have been observed with the drug. Although numerous
studies carried out in malaria endemic countries had shown good
efficacy and safety of ACT for the treatment of uncomplicated
malaria, the conditions of clinical trials do not fully reflect real
field situation. Results from studies conducted with unsupervised
malaria treatment showed low cure rate after adjustment by day
28 [16,18,19,23].ASAQ was well-tolerated, similar to many other
studies with AEs mostly mild and not linked to the administrated
treatment [20,17,11].Safety and tolerability monitoring of ASAQ
and other forms of ACT should continue in a standardized
manner. Unfortunately pharmacovigilance networks are not
implemented in most settings where ACT is routinely used.
This therapeutic efficacy study had several limitations. First
this study of drug efficacy have limited follow-up to 28 days, the
minimum recommended by WHO, and thus only the short-term
effectiveness has been assessed . Any additional recurrences
beyond this time frame were not captured (42 days). Secondly
drug levels were not tested and challenge of getting reliable
safety recall information from children our study population. The
results of this study demonstrate that ASAQ remain efficacious
treatments for uncomplicated P. falciparum malaria in Chad.
There is no evidence at this time that a change in regimens is
warranted. However, continued monitoring of drug efficacy,
following WHO recommendations, is needed. In the bulk of the
sub-Saharan African countries, malaria drug policies relay mostly
on the use of the artemisinin combination drugs treatment. To
avoid the pitfall known with chloroquine and to preserve as long
as possible the effectiveness of these ACTs, a better understanding
of the underlying mechanisms associated with resistance or loss
of susceptibility to these combinations is necessary to ensure an
optimal use . Thus, in the present study, the DNA of 58 isolates
of P. falciparum was analyzed to check mutations associated with
resistance to the ACTs. As previously reported and in line with
some previous work implemented within sub-Saharan Africa, no
parasite of the analyzed sample harbored any mutation neither
synonymous nor non-synonymous. This result is in conformity
with the clinical data with no records of any therapeutic failure.
Our result is similar to that reported in Benin and India where
the analysis of Kelch 13-propeller sequences indicated that all
isolates were of wild type [20-22]. All were no synonymous
mutations . The absence of mutation in the gene k13-
propeller in our study could explain the sensitivity of the parasite
to ASAQ. However, the results of our study could not be inferred
to the whole country, even to the Western Mayo-Kebbi region,
since only one Pala site hosted the study. The geographical and
epidemiologic settings within this Western Mayo-Kebbi region
differed from one point to another. Thus, additional studies are
needed in order to increase samples size for robust analysis and
relevant resistant data from Chad.
Artesunate–Amodiaquine (ASAQ) has been shown to be
safe and highly effective in the treatment of uncomplicated
P. falciparum malaria for children in Pala. No evidence of the
emergence of artemisinin resistance in Tchad was found upon
investigation of mutations in the k13 propeller domain. However,
additional clinical and molecular studies need to be performed in
different parts of the country to provide clear and relevant data
related to drug resistance in Chad.
This study was supported financilay by the Bill & Melinda
Gates Foundation through WHO and implemented by the NPME
with the support of the Minister of health.
Conflict of interest
The authors state that there is no conflict of interest.
DH and PR are staff members of the World Health Organization.
DH and PR alone are responsible for the views expressed in this
publication and they do not necessarily represent the decisions,
policy or views of the World Health Organization.
The procedures followed were in accordance with the ethical
standards of Helsinki Declaration. Consent form was signed by
parent or legal guardian of children included in the study.
Approbation was given by the Ministry of Public Health under
ethical clearance N° 2192/PR/PM/MSP/SE/SG/DGAS/DSPELM/
DMTNT/PNLP/15 and by WHO ERC.
ASAQ: Artesunate-Amodiaquine; SP: Sulfadoxine-
Pyrimethamine; AL: Artemether-Lumefantrine; TBS: Thick
Blood Smear ; NPME: National Program For Malaria Eradication
In Chad; MPH: Ministry Of Public Health; DS: Sanitary District;
SNPs: Single Nucleotide Polymorphisms; IPCI: Institut Pasteur
Of Côte d’Ivoire; NCBT: National Center For Blood Transfusion;
WHO: World Health Organization; PALAT: Support Project For
Malaria Control In Chad; UNDP: United Nations Development
Program; GMP: Global Malaria Program; ACT: Artemisinin-
Based Combination Therapy; ACPR: Adequate Clinical And
Parasitological Response; LPF: Late Parasitological Failure; LCF:
Late Clinical Failure; ETF: Early Therapeutic Failure; LPV: Lost
Patient View; PRS: Patient Removed From Study; MPD: Mean
Parasite Density; DP: Parasite Density; CI: Confidence Index; Ds:
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