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Artemisinins resistance molecular markers in falciparum malaria in some sentinel site of Central Highland Vietnam
Huynh Hong Quang et al., 1 1Institute of Malariology, Parasitology, and Entomology Quy Nhon, MoH Abstract Introduction: Malaria is a vector-borne disease by far the world's most important tropical parasitic disease with high mortality and morbidity, especially in tropical Africa and South East Asia. The emergence of artemisinin resistance in Vietnam as big obstacle to reduce the malaria burden and elimination. Methods: This study conducted both therapeutic efficacy in vivo test and PCR-sequencing for genotyping SNPs PF3D7_1343700 Kelch protein propeller domain in Plasmodium falciparum K13 locus. Results: these data showed that a correlation between parasite clearance and predominantly K13 mutation of C580Y with 22/29 cases (75.86%) in Gia Lai and 3/20 cases (15%) in Ninh Thuan sentinel sites in ACTs-treated patients. These mutations conferred elevated resistance to recent Cambodia, Laos, and Myanmar isolates compared with that of reference lines. Conclusion: These data provide a conclusive rationale for Central Vietnam K13-propeller sequencing to identify artemisinin-resistant falciparum parasites, but their importance in artemisinin resistance remains to be elucidated. Key words: Plasmodium falciparum, artemisinin resistance, K13 propeller mutation 1. BACKGROUND The Plasmodium falciparumparasitehas a remarkable capacity to develop resistance to antimalarial drugs by evolutionary adaptation. Artemisinin-based combination therapies (ACTs) are now recommended by the World Health Organization as first-line treatment for uncomplicated falciparum malaria. However, clinical resistance to artemisinin and its derivatives is now well established in theP. falciparum population of Cambodia, Myanmar, Laos and appears to be emerging in neighbouring regions [1],[2],[11]. The proportion of patients who are parasitemic on day 3, which is currently the indicator of choice for routine monitoring to identify suspected artemisinins resistance in P.falciparum. Furrthermore, PF3D7_1343700Kelch protein propeller polymorphism shows a significant association with RSA0-3?hsurvival rates [7], simultaneously matches that of slow parasite clearance which reflects the reduced susceptibility of ring stage parasites and the day 3 positivity in ACTs-treated patients [3]. As a result, K13-propeller polymorphism fulfils the definition of a molecular marker of artemisinine resistance.Consequently, there is an urgently need to discover the parasite genetic factors that cause artemisinin resistance and to identify potential markers to monitor its spread [4]. Therefore, this research of artemisinin resistance markers infalciparummalaria in Central Vietnam with following objectives: 1. To identify the prevalence of delayed parasite clearance and the frequency of single nucleotide polymorphism (SNPs) K13 propeller in malaria endemic areas; 2. To investigate the correlation between two markers (delayed parasite clearance and SNPs K13 polymorphism) and clinical artemisinin resistance in falciparum malaria. 2. METHODOLOGY 2.1.Locations and timeframe -The study was conducted in multi-centers in malarial hyperendemic areas at Krong Pa district (Gialai province), and Ninh Son district (Ninh Thuan province); -From January 2015 to May 2016. 2.2.Study methods -Study design with modified fast 7-day in vivo test protocol, Non randomised controlled study; -Sample size: with classical statistical methods are recommended for determining sample size, on the basis of an expected proportion of treatment failures, desired confidence interval (CI 95%) and precision (10%). In the case of a medicine with an expected failure rate of 10%, a CI 95% and a precision level of 10%, a minimum of 35 patients should be enrolled. Table 1. Calculation of sample size in vivo test Estimated population proportion (p), confidence interval 95% | d | 0.05 | 0.10 | 0.15 | 0.20 | 0.25 | 0.30 | 0.35 | 0.40 | 0.45 | 0.50 | | 73 | 138 | 196 | 246 | 288 | 323 | 350 | 369 | 380 | 384 | 0.10 | 18 | 35 | 49 | 61 | 72 | 81 | 87 | 92 | 95 | 96 |
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2.3.Patients and materials Inclusion criteria +Age between 2 to 70 years old; +Mono-infection with P. falciparum detected by light microscopy; +Parasitaemia of500 - 100.000 asexual forms/µl blood; +Presence of axillary temperature ? 37.5°C or history of fever (past 24h); +Ability to swallow oral medication; +Ability and willingness to comply with the study protocol for the duration of the study and to comply with the study visit schedule; +Not yet take any antimalarial drugs; +Informed consent from the patient or parents in the case of children; Exclusion criteria +Age under 2 years or more than 70 years old; +Presence of general danger signs or signs of severe falciparum malaria; +Mixed or mono-infection with another Plasmodium species; +Presence of severe malnutrition, febrile conditions due to diseases other than malaria (acute lower respiratory tract infection, severe diarrhoea) or other known underlying chronic or severe diseases, severely vomitting, or psychological disorders; +History of hypersensitivity reactions or contraindications to any of the medicine(s) being tested; +A positive pregnancy test or breastfeeding women; 2.4. Antimalarial drugs to be tested in clinical trials Dihydroartemisinin-piperaquin phosphate (DHA-PPQ) tablet, dosage regimen as followed by latest national guidelines for malaria diagnosis and treatment (MoH, 2013). 2.5. Sampling and techniques P. falciparum isolates were obtained from patients at calendar?s days after treatment with DHA-PPQ. Thick and thin films were stained, and the parasite densities were determined by counting the number of asexual parasites per 200 white blood cells (WBCs), assuming a WBCs count of 8.000/µl. A slide was considered negative if no asexual parasites were found after counting 1,000 WBCs. Patients will be treated with DHA-PPQ and their parasite density will be measured every 12h until parasitaemia is undetectable. Sampling capillary blood was collected by finger prick and stored in EDTA tubes; All of standard operational procedures (SOPs) for DNA extraction, PCR, nested-PCR, quantitative parasite load by qPCR at day 0 (D0), day 3 (D3), real-time PCR amplification and standard curve using serial dilutions of DNA from cultured parasites, PCR-Sequencing for genotyping SNPs PF3D7-1343700 Kelch protein propeller domain, agarose gel electrophoresis, and genetic factors analysis conditions followed by Ariey's protocol (2014), CFX 96 and ApoE softwares [3],[7]. 2.6. Ethical approval Ethical clearances for this study from patients will be obtained from the Institutional Review Board of the Institute of Malariology, Parasitology, and Entomology (IMPE) Quy Nhon and Hue University of Medicine and Pharmacy. Work will be conducted in compliance with all relevant ethical standards and regulations governing research involving human subjects. 3. RESULTS 3.1.The trial profile and baseline characteristics of patients Of the 56 falciparum malaria patients screened, males (64.3%) dominantly outnumbered females, and all most were in group more than 15 years old (87.1% and 88%). Seven patients (12.5%) did not fulfill the entry criteria, lost follow-up after two days treatment or lost the samples on D0 or D3, resulting in 49 (87.5%) patients recruited. Thirsty-three (67.3%) patients completed the 3-day course of DHA-PPQ, and 16 (32.7%) patients completed the 7-day follow-up. Table 2. Baseline characterization of the patients, according to study site Study site | No. cases | Male sex | Median age | Median parasitemia by PCR on D0 | Median parasitemia by smear on D0 | Gameto -cytemia on D0 | Mean hematocrit (%) | Gia Lai | 29 | 24 (77.42%) | 26 | 14.150 (43-825.017) | 33079 (1.409-135.250) | 5 (17.24%) | 37.93 (40.41-2.12) | Ninh Thuan | 20 | 12 (48%) | 29 | 9038 (140-625.012) | 10.833 (2.388-93.755) | 2 (10%) | 40 (37.9-44.85) | Total | 49 | 36 (73.47%) | 25 | 12.125 (43-825.016) | 26.914 (1.409-135.250) | 7 (14.3%) | 38.77 (37.9-44.85) |
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The median malaria parasite density of asexual P. falciparum at two sentinel sites were around 12.125 (43-825.016)/mL by PCR and 26.914 (1.409-135.250)/µL by microscopy. Number of cases with positive gametocyte were from 10-17.24% in Gia Lai, and Ninh Thuan, respectively. Table 3. Baseline clinical patient's profile at the D0 Studied patient's profile | At the point start of study D0 | Gia Lai (n = 29 ) | Ninh Thuan (n = 20) | Total (N = 49) | Temperature & body weight Mean temperature in 0C Mean weight in kg Fever day number before test | 37.78 ± 1.2 36.5 ± 18.2 2.6 ± 1.1 | 38.16 ± 1.0 39.5 ± 15 2.2 ± 1.2 | 38.19 ± 1.0 38.5 ± 12.0 2.4 ± 1.2 | Fever or history of fever Body temperature ? 37.50C History of fever(past 48 hours) | 23 (79.3%) 3 (10.34%) | 14(70%) 3(15%) | 37 (75.5%) 6 (12.24%) |
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Only 12.24% of patients, the onset of fever occurred within the previous 72 h, and 75.5% had measurable fever at the time of recruitment. 3.2.The parasite clearance rate Table 4. The efficacy of DHA-PPQ in parasite clearance Malaria patients | Total (N = 49) | Gia Lai (n = 29) | Ninh Thuan (n = 20) | PCR | % | Smear | % | PCR | % | Smear | % | PCR | % | Smear | % | No DPC | 16 | 32.7 | 39 | 79.6 | 4 | 13.8 | 20 | 69 | 12 | 60 | 19 | 95 | DPC | 33 | 67.3 | 10 | 20.4 | 25 | 86.2 | 9 | 31 | 8 | 40 | 1 | 5 |
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The parasite clearance rate was assessed in 49 patients. Number of non-delayed parasite clearance_DPC) rate in general were obviously different by qPCR and by microscopy, 32.7% and 79.6%, respectively or DPC of 67.3%by qPCR and 20.4% by microscopy.  | Fig 1. Distribution of malaria patients according to their age |
The highlights of the DPC, or proportion of positive asexual P. falciparum atD3 rate in Gia Lai was extremely high with 86.2% and 40% in Ninh Thuan. With in total 49 patients, 16 (32.7%) patients completed the 28-day follow-up, there were 9 (56.3%) cases were positive at Dq28 by qPCR with low parasitemia (from 1- 43 parasites/µl).
Analysis of all 33 cases with parasite positive on D3 (t72), 7 (21.2%) patients had the residual parasitemia after 72 h was more than 1% (severe DPC), and all the other patients with DPC, residual parasitemia was less than 1 % (Mild DPC). Age distributions followed No DPC, Mild DPC and Servere DPC have no significantly difference (Fig. 1).  | Fig2. In parasite clearance 72 hours after teatment (t72), samples have been grouped according to the residual parasitemia at t72:
Samples without measurable parasitemia at time 72h (No DPC), samples with residual parasitemia <1% (Mild DPC), and samples with
residual parasitemia >1% (Severe DPC). |
3.2.1. Delayed parasite clearance in Gia Lai
Totally 29 cases in Gia Lai, the highlights of the DPC, or proportion of positive asexual P. falciparum at D3 rate in Gia Lai was extremely high with 86.2% by qPCR, even though very high rate 31% by microscopy. Within Fig 2, there were 6 cases which were severe DPC. They also located in Gia Lai (Fig 2- Severe DPC in Gia Lai). Another 19 cases were in Mild DPC, just only 4 cases were No DPC.  | Fig 3. Parasite clearence 72 hours after teatment (t72) with DHA-PPQ in Gia Lai |
3.2.2.Delayed parasite clearance in Ninh Thuan  | Fig 4. Parasite clearance 72 hours after teatment (t72)withDHA-PPQ in Ninh Thuan |
Almost No DPC cases within our research located in Ninh Thuan was confirmed in this site. In addition to, there were the others of 6 cases were in mild DPC.
3.3.Nonsynonymous SNPs Kelch 13 polymorphisms observed in P. falciparum isolates 3.3.2.Result of Nested PCP of K13 gene in agarose gel electrophoresis  | Fig 5.PCR products for Secondary N1 PCRfor K13 gene S1: tested samples, Neg: PCR negative controls M: 1000bp marker; Expected size of PCR product: 849 bp |
3.3.3.Nonsynonymous SNPs Kelch-13 polymorphisms observed in the P. falciparum isolates Table 5. Polymorphisms observed in the K13-propeller P. falciparum isolates Study site | No.of patients / Total no. (%) | Codon position | Amino Acid reference | Nucleotide reference | Amino Acid Mutation | Nucleotide Mutation | Gia Lai (n= 29) | 22 (75.86%) | 580 | C | TGT | Y | TAT | Ninh Thuan (n=20) | 3 (15%) | 580 | C | TGT | Y | TAT | Total (n= 49) | 25 (51 %) | 580 | C | TGT | Y | TAT |
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A total of 49 collected samples were examined for the SNPs of the K13 propeller domain. Just only C580Y was obtained. The overall prevalence of the mutant allele was 51% (25/49). However, the mutant allele obtained were in Gia Lai with 75.86% (22/29), meanwhile, the prevalence of C580Y in Ninh Thuan was 15% (3/20).
In comparison with classification of K13 polymorphism (Followed by WHO, 2015) K13 mutant alleles C580Y observed in two province, Gia Lai and Ninh Thuan was reported indicate emergingartemisinin resistance. It is one of confirmed K13 propellermutations(i.e. those confirmed by in vivo and in vitro data). 3.4.The correlation between the K13 polymorphisms and delayed parasite clearance 3.4.1.Correlation between the frequency ofK13-propeller alleles and the prevalence of D3(+) Table 6. Comparison with classification of K13 polymorphism with WHO reference (2015)
Table 7. The frequency of K13 mutants correlated to the prevalence of DPC
No. | The frequency / the prevalence | Province | Total (n = 49) | Gia Lai (n = 29) | Ninh Thuan (n = 20) | n | % | n | % | n | % | 1 | Wild-type K13-propeller alleles | 7 | 24.14 | 17 | 85 | 24 | 48.98 | 2 | Mutant K13-propeller alleles | 22 | 75.86 | 3 | 15 | 25 | 51.02 | 3 | DPC with mutant K13-propeller alleles | 20 | 90.9 | 2 | 9.1 | 22 | 44.9 | 4 | DPC with wild-type K13-propeller alleles | 5 | 17.24 | 5 | 25 | 10 | 20.4 | 5 | No DPC with mutant K13 alleles | 2 | 6.89 | 1 | 5 | 3 | 6.12 | 6 | No DPC with wild-type K13 alleles | 2 | 6.89 | 12 | 55 | 14 | 28.58 |
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All of 49 patients, 25 (51.02%) cases had mutant K13 propeller alleles, mostly 44.9% (22/49) were positive of parasite on day 3 after treatment, just only 3 cases had K13 mutant (6.12%) were negative with DPC. And mostly cases of DPC with K13 mutant occurred in Gia Lai in 90.9 % (20/22). Very few patients of positive DPC with K13 mutant were obtained in Ninh Thuan in 9.1% (2/22). |  | Fig 6. The number of cases positive with delayed parasite clearance and K13 mutant alleles |
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The presence of C580Y mutant was obtained mostly in the mild DPC group, followed by severe DPC group. In general, the number cases of mild DPC and severe DPC correlated with the frequency of K13 propeller C580Y. Almost patients have no delayed parasite clearance were obtained with wild type K13 mutant alleles. The sensitivity and specificity of the SNP 580Y at day 0 to identify delayed clearance (PCT, ? 72h) were therefore estimated at 71.88% (22/32) and 82.35% (14/17), respectively; the positive predictive value of the mutation was 88% (22/25), while the negative predictive value of the wild-type allele was 58.33% (14/24). The cases positive with K13 mutation and also DPC mostly occurred in Gia Lai.3.4.2. Correlation between the frequency ofK13-propeller alleles and the proportion D3 (+) The mutant allele obtained were in Gia Lai with extremely high prevalence 75.86% (22/29), compared to the frequency of C580Y in Ninh Thuan was 15% (3/20). 22 cases were positive of K13 mutantation and DPC, mostly occurred in Gia Lai,just2 cases were obtained in Ninh Thuan. Within a large number of patients had the C580Y allele (25/49), all of 6 patients of severe DPC group had C580Y mutation were obtained in Gia Lai. No patient of severe DPC with C580Y was obtained in Ninh Thuan. Just only 2 patients had C580Y allele were found in mild DPC group and the other one in No DPC group. All most No DPC patients with K13 wild type allele were found in Ninh Thuan, 12 in 14 totally. 3.4.3.Correlation between the frequency ofK13-propeller alleles and several cases were positive at D28 after ACT treatment  | Fig 7. The number of cases positive with delayed parasite clearance and K13 mutant alleles |
Within totally 49 patients, 16 (32.7%) patients completed the 28-day follow-up, there were 9 (56.3%) cases were positive at D28 by qPCR with low parasitemia (from 1-43 parasites/µl). Just only 2 cases in Gia Lai were confirmed to carry K13 mutant allele C580Y.
4. DISCUSSION 4.1.Baseline characterization of the patients and efficacy of DHA-PPQ in parasite clearance All of 49 patients with the median malaria parasite density of asexual P. falciparum at two sites Gia Lai and Ninh Thuan were 14.150 and 9.038 parasites/µ respectively. The low median parasitemia in two provinces are not an effect to be considered as the factor contributing to delay in parasite clearance [5],[6]. Compared to microscopy, the prevalence of parasite densities by qPCR detected increasingly more infections over time, and the difference became significant at day 3, when the prevalence by PCR was 3.3 times higher than that by microscopy (67.3% vs. 20.4%; p < 0.01). Notably, the proportion of positive parasitemia at D3 in Gia Lai province was extremely high, 86.2% by qPCR and 31 % by microscopy; in Ninh Thuan was 40% by qPCR and 5% by microscopy, which is currently the indicator of choice for routine monitoring to identify suspected artemisinin resistance in P. falciparum.
Using qPCR with high sensitivity and specificity, the number of positive parasitemia on D3 observed by qPCR much higher in comparing with microscopic evaluation. However, within the large range of parasitemia on D3 observed by qPCR, we divided all 33 delayed parasite clearance cases into two group mild DPC (with residual parasitemia <1%) and severe DPC (residual parasitemia >1%). In the mild DPC group, the parasitemia was low, from 1 to less 10 parasites/µl. No evidence showed that the very low parasitemia detected by qPCR probably was death parasite or not. Almost mild DPC cases focussed in Gia Lai and in contrast, No DPC cases focussed in Ninh Thuan mainly, and it is necessary to have further study about it.
About the DHA-PPQ efficacy in Gia Lai, specially, there were just only 6 cases in severe DPC group which located in Gia Lai, where the median parasitemia and the prevalence of D3 positive after ACTs treatment were confirmed to be higher than in Ninh Thuan. High parasitemia on D3 after using DHA-PPQ in severe DPC group is unlikely that was the immunity profile cause of the observed delayed parasite clearance. It is necessary to complete futher study about the immunity profile of the community and the individual, also the pharmacokinetics and pharmacodynamics of the antimalarial drug to understand this situation. High prevalence of positive parasitemia on D3 was also similar to the other sites in the Gia Lai, Dak Nong, Quang Nam [8], Khanh Hoa, Binh Phuoc [5],[6]. The the proportion of positive parasitemia at D3 in Binh Phuoc was from 15% up to 22%; 30%; 36%, and 36.8% year by year from 2010 to 2015, in Quang Nam of 29.2%, Kon Tum of 14%, Khanh Hoa of 17.4%, Dak Nong was from 29% up to 26.7% from 2012 to 2014, and Gia Lai was from 11% up to 23%; 26.4% and 38.5% from 2010 to 2014. Not only Vietnam but also the other countries in South East Asia, the the proportion of positive parasitemia at D3 in Cambodia increased continuously year by year 26% up to 33%; 45% and 54% from 2008 to 2012, in Myanmar from 14% up to 19%; 23% from 2012 to 2013 or Thailand was from 9% up to 14%; 17%; 25% and 48% from 2009 to 2014. About the DHA-PPQ efficacy, with 16 cases followed up to D28, the adequate clinical and parasitological response (100% in Ninh Thu?n and 97.4% in Gia Lai), negative parasitaemia on D28 was 56.3% (9/16) confirmed by qPCR, divided into two province equally. And all of cases were positive at D28 had the proportion of positive asexual P. falciparum at D3 by qPCR. This result is different to previous researchs of Vietnam [5],[6]. It probably caused by using different sensitivity methods to detect parasitemia. It is necessary to to apply with the other samples to assess the DHA-PPQ efficacy. Moreover, age distributions followed No DPC, mild DPC and servere DPC have no significantly difference (Fig 12). As expected from previous work, young children had slower parasite clearance rates compared to older patients. However, this was observed only in artemisinin-sensitive parasite populations, with most data coming from Africa. Resistant parasite populations, present only in Southeast Asia, did not demonstrate an age effect. The lack of an age effect on DPC could be due to one or more of the following factors: lower background immunity in those patients from low transmission settings, different age distributions studied with 87.5% of patients aged older than 15 years. 4.2.Frequency of non synonymous Kelch 13 polymorphisms observed in P. falciparum isolates In this study, the overall prevalence of the mutant allele was 51% (25/49). And the C580Y mutant allele was only one SNPs K13 propeller found in all cases positive with K13 mutation. Notably, the C580Y substitutions that was confirmed to correlate to in vitro resistance or delayed parasite clearance - a confirmed mutation alleles inclassification of K13 polymorphism as WHO reference in Southeast Asia.About the correlation between the K13 polymorphisms and delayed parasite clearance, the presence of C580Y mutant was obtained mostly in the mild DPC group, followed by severe DPC group. In addition, almost no DPC cases had wild-type K13 mutant allele. In general, the number cases of mild DPC and severe DPC correlated with the frequency of K13 propeller C580Y. However, the mutant allele obtained were in Gia Lai with extremely high prevalence compared to the prevalence of C580Y in Ninh Thuan (75.86% vs. 15%). The cases positive with K13 mutation and also DPC mostly occurred in Gia Lai. Furthermore, C580Y K13 alleles mainly were obtained in mild DPC group. This data gave the correlation between the delayed parasite clearance and the K13 mutation in this sites (p = 0.002). on the contrary, in Ninh Thuan, no DPC showed a relevent data to K13 wild-type. Considering that the high prevalence of delayed parasite clearance and K13 mutant alleles, Gia lai fulfills the WHO definition of confirmed artemisinin resistance and Ninh Thuan as suspected artemisinin resistance. 4.4. The correlation between the K13 mutations, DPC, and artermisinin resistant P. falciparum The frequency distribution of mutant alleles over two provinces matches that of day 3 positivity in ACTs-treated patients. The mutant parasites cluster in Gia Lai provinces where the delayed parasite clearance is well established and are less prevalent where DPC is less common in Ninh Thuan. The study was designed as a 28-day follow-up study to assess the efficacy of DHA-PPQ in treating falciparum malaria. Despite the fact that no recrudescent case was obtained during the researching time, there were 9 cases were positive at D28 by qPCR with low parasitemia. The hypothesis of death parasite could be detected until D28. It also means that the parasite could be still alive until after day 20 at least. Within 9 cases were positive of asexual parasite on D28, two of them also carried C580Y mutant alleles. It seems to be a strong evidence of the correlation between K13 mutation and delayed parasite clearance [9]. Nevertheless, no evidence of rescrudescence was showed. It leads to the other hypothesis that the patiens with positive of parasite on D28 could become a asymptomatic carrier [10]. In study, the frequency of mutant alleles correlates strongly with the prevalence of D3 positivity after ACT treatment; however, both K13 mutant alleles and DPC were not associated with artemisinin resistance. In fact, totalof186K13alleles,including108non-synonymousmutations havebeenreporteduntil now. However, notallnon-synonymouspropeller-regionK13 mutantsreportedindicateemergingartemisinin resistance. Besides, the emergence of SNPs K13 propeller and the spread of artemisinin seem to be independent. Notebly, the widespread 580Y mutation seems to have emerged twice. Given that frequency distribution suggests a Cambodian origin, where the mutations are most prevalent, with subsequent spread to Vietnam and maybe to Africa [9]. Howerver, our study site in Krong Pa, Gia Lai does not share the border with Cambodia, therefore, C580Y allele maybe one of indigenous mutation. It created ?the gap, the blacklash? on Tier 1 map on Central highland of Vietnam. Although few P. falciparum isolates were recruited in Ninh Thuan, it shows the high prevalence of DPC with low frequency of K13 mutations, it is indispensable to continue further study to assess the DHA-PPq efficacy for P. falciparum infection in this area and interprete the role of K13 mutant alleles in this area. CONCLUSION Up-to-date information on whether artemisinin resistance has already disseminated or independently emerged is a critical issue. If resistance were limited to a small, well-defined area, then elimination of drug-resistant malaria from these regions would be possible. More works are needed to delineate the normal function of K13 and the effect of various mutations besides studies are therefore required to identify additional genetic determinants of artemisinin resistance. Conflict of interest The authors have no other conflicts of interest. Acknowledment This study is partly supported by the donations of World Health Organization (WHO), Sassari University, Institute of Malariology, Parasitology, and Entomology (IMPE) Quy Nhon, Vietnam and Hue University of Medicine and Pharmacy. REFERENCES 1.Boullé M, Witkowski B, Valentine Duru, Kanlaya Sriprawat, Nair SK, McDew-White M, Anderson TJC, Phyo AP, Menard D, Nosten F (2016). Artemisinin-resistant Plasmodium falciparum K13 mutant alleles, Thailand-Myanmar Border. Emerging Infectious Diseases, 22(8):1503-1055. 2.Amaratunga C, Sreng S, Suon S, Phelps ES, Stepniewska K, Lim P, et al (2012). Artemisinin-resistant Plasmodium falciparum in Pursat province, western Cambodia: a parasite clearance rate study. 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