Research Article Open Access
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
*Corresponding author: 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.
Abstract Top
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 sequence PF3D7_1343700.

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 resistance surveillance.

Key Word: Malaria, P. falciparum, Artesunate-Amodiaquine, k13- propeller, Chad
Introduction
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 [1-5].

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 [7]. 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 populations [8].

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.
Methods
Study design
This study was a prospective, one-arm assessment of clinical and tolerability of ASAQ according to WHO guidelines [8]. 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 [12].
Study population
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 [8].
Study treatments
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.
Follow-up procedure
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 also recorded.

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 and recrudescence.
Safety
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.
Outcomes
Treatments outcomes were classified based on clinically and parasitological outcomes assessment as recommended by WHO [8].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].
Statistical analysis
Data management and analysis were completed with Epi Info 6.0.4 adaptedc to the Who excel-based applications [7].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.
Results
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

Parameters

Items

Screened

163

Included n (%)

58 (35.6 %)

Male n (%)

26 (44.8 %)

Female n (%)

32 (55.2 %)

Sex ratio

0.81

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)

3840

Parasitemia (min - max) asexual parasites / μL

1.040 - 20.000

Therapeutic efficacy
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

 

Per-protocolanalysis

Patients screenedat D28

51 (87.9%)

Late clinical failure

1 (2%)

Adequate Clinical and Parasitological Response at D28

50 (98%)

Adequate Clinical and Parasitological Response at D28 after PCR

51 (100%)

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 detectable polymorphism.
Discussion
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 [11]. 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, [13]. 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 [14]. 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 [24]. 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 [14]. 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 [25]. 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.
Conclusion
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.
Acknowledgements
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.
Ethics
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.
Abbreviations
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: Standard Deviation.
ReferencesTop
  1. Noedl H, Se Y, Schaecher K, Smith BL, Socheat D and Fukuda MM. Evidence of artemisinin resistance in western Cambodia.        N Engl J Med. 2008;359(24):2619–2620. doi:10.1056/NEJMc0805011
  2. Noedl H, Se Y, Sriwichai S, Schaecher K, Teja-Isavadharm P, Smith B. Artemisinin resistance in Cambodia: a clinical trial designed to address an emerging problem in Southeast Asia. Clin Infect Dis. 2010;51(11):82–89. doi:10.1086/657120
  3. Carrara VI, Zwang J, Ashley EA, Price RN, Stepniewska K, Barends M, et al. Changes in the treatment responses to artesunate-mefloquine on the northwestern border of Thailand during 13 years of continuous deployment. PLoS One. 2009;4(2):e4511. doi:10.1371/journal.pone.0004551
  4. Lim P, Alker AP, Khim N, Shah NK, Incardona S, Doung S, et al. Pfmdr1 copy number and arteminisin derivatives combination therapy failure in falciparum malaria in Cambodia. Malar J. 2009;8(11). doi:10.1186/1475-2875-8-11.
  5. Phyo AP, Nkhoma S, Stepniewska K, Ashley EA, Nair S, McGready R. Emergence of artemisinin-resistant malaria on the western border of Thailand: a longitudinal study. Lancet. 2012;379(9830):1960–1966. doi:10.1016/S0140-6736(12)60484-X
  6. Ariey F, Witkowski B, Amaratunga C, Beghain J, Langlois AC, Khim N, et al. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature. 2014;505(7481):50-55. doi:10.1038/nature12876.
  7. Ashley EA, Dhorda M, Fairhurst RM, Amaratunga C, Lim P, Suon S, et al. Spread of Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med. 2014;371:411–423. doi:10.1056/NEJMoa1314981
  8. WHO. Methods for surveillance of antimalarial drug efficacy. Geneva: World Health Organization. 2009:85
  9. Chatterie M, Ganguly S, Saha P, Bankura B, Basu N, Das M, et al. No polymorphism in Plasmodium falciparum K13 propeller gene in clinical isolates from Kolkata. India. J Pathog. 2015;2015(374354). doi:10.1155/2015/374354
  10. WHO. Susceptibility of Plasmodium falciparum to antimalarial drugs: report on global monitoring: 1996-2004. Geneva: World Health Organisation. 2005;133.WHO/HTM/MAL/.1103
  11. Oguche S, Okafor HU, Watila I, Meremikwu M, Agomo P, Ogala W, et al. Efficacy of artemisinin-based combination treatments of uncomplicated falciparum malaria in under-five year-old Nigerian children. Am J Trop Med Hyg. 2014;91(5):925–935.            doi:10.4269/ajtmh.13-0248
  12. Ariey F, Witkowski B, Amaratunga C, Beghain J, Langlois A-C, Khim N, et al. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria. Nature. 2013;505(7481):50–55. doi:10.1038/nature12876
  13. Falade CO, Dada-Adegbola HO, Ogunkunle OO, Oguike MC, Nash O, Ademowo OG. Evaluation of the comparative efficacy and safety of artemether-lumefantrine, artesunate-amodiaquine and artesunate-amodiaquine-chlorpheniramine (Artemoclo™) for the treatment of acute uncomplicated malaria in Nigerian children. Med Princ Pract. 2014;23(3):204-211.                                     doi:10.1159/000360578
  14. Djallé D, Njuimo SP, Manirakiza A, Laganier A, Le Faou A and Rogier. Efficacy and safety of artemether + lumefantrine, artesunate + sulphamethoxypyrazine-pyrimethamine and artesunate + amodiaquine and sulphadoxinepyrimethamine + amodiaquine in the treatment of uncomplicated falciparum malaria in Bangui, Central African Republic: a randomized trial. Malaria Journal. 2014;13:9. doi:10.1186/1475-2875-13-9
  15. Sondo P, Derra K, Diallo-Nakanabo S, Tarnagda Z, Zampa O, Kazienga A, et al. Effectiveness and safety of artemether-lumefantrine versus artesunate-amodiaquine for unsupervised treatment of uncomplicated falciparum malaria in patients of all age groups in Nanoro, Burkina Faso: a randomized open label trial. Malaria Journal. 2015;14:325.doi:10.1186/s12936-015-0843-8
  16. Tinto H, Diallo S, Zongo I, Guiraud I, Valea I, Kazienga A, et al. Effectiveness of artesunate–amodiaquine vs. artemether–lumefantrine for the treatment of uncomplicated falciparum malaria in Nanoro, Burkina Faso a noninferiorityrandomised trial. Trop Med Int Health. 2014;19(4):469-475. doi:10.1111/tmi.12274
  17. Lise MUSSET. Contribution a l'étude de la résistance Plasmodium falciparum à l'atovaquone-proguanil. 2006.
  18. Sowunmi A, Akano K, Ntadom G, Ayede AI, Ibironke FO, Aderoyeje T, et al. Therapeutic efficacy and effects of artemisinin-based combination treatments on uncomplicated Plasmodium falciparum malaria -associated anaemia in Nigerian children during seven years of adoption as first-line treatments. Infectious Diseases of Poverty. 2017;6:36.
  19. Zenglei W, Sony S, Xiaolian L, Jun M, Lili Y, Mynthia C, et al. Prevalence of K13-propeller polymorphisms in Plasmodium falciparum from China-Myanmar border in 2007–2012. Malaria Journal. 2015;14:168. doi:10.1186/s12936-015-0672-9
  20. Aurore O, Georgia D, Awa BD, Nicaise TN, Constance A, Didier T, et al.  Lack of artemisinin resistance in Plasmodium falciparum in northwest Benin after 10 years of use of artemisinin-based combination therapy. Parasite. 2016;23:28.                                            doi:10.1051/parasite/2016028
  21. WHO/Communicable diseases cluster. Severe falciparum malaria. Trans R Soc Trop Med Hyg. 2000;94:51–90
  22. World Health Organization. Status report on artemisinin and ACT resistance. Geneva: World Health Organization 2015.
  23. Offianan A Toure,Serge B Assi, Tiacoh L N’Guessan, Gbessi E Adji, Aristide B Ako, Marie J Brou, et al. Open-label, randomized, non-inferiority clinical trial of artesunate-amodiaquine versus artemether-lumefantrine fixed-dose combinations in children and adults with uncomplicated falciparum malaria in Côte d'Ivoire. Malaria Journal. 2014;13:439. doi:10.1186/1475-2875-13-439
  24. WHO. Methods for surveillance of antimalarial drug efficacy. Geneva: World Health Organization. 2009;85.
  25. Ménard D,  Khim J, Beghain AA, Adegnika M, Shafiul‑Alam O, Amodu G, et al. : A Worldwide Map of Plasmodium falciparum K13-Propeller Polymorphisms, for the KARMA Consortium. New England Journal of Medicine. 2016;374(25):2453-2464.         doi:10.1056/NEJMoa1513137
 
Listing : ICMJE   

Creative Commons License Open Access by Symbiosis is licensed under a Creative Commons Attribution 3.0 Unported License