Researches progress on molecular surveillance of artemisinin resistance in Plasmodium falciparum

doi:10.12140/j.issn.1000-7423.2025.02.017

Abstract

Abstract:

The widespread deployment of artemisinin-based combination therapies (ACTs) for falciparum malaria has led to emergence of partial resistance to artemisinin and its derivatives, which has become one of the greatest challenge in the global malaria control programme. Recently, great strides have been achieved in unraveling the molecular mechanism underlying the resistance of P. falciparum to artemisinin and identification of molecular markers. The currently identified P. falciparum artemisinin resistance-associated genes include kelch protein 13 (pfk13), ubiquitin carboxyl-terminal hydrolase 1 (pfubp1), AP-2 complex subunit mu (pfap2μ), and coronin (pfcoronin). Molecular surveillance data reveal widespread distribution of P. falciparum artemisinin resistance-associated gene mutations across Southeast Asia, and emergence of artemisinin-resistant P. falciparum strains of independent origin in Africa, South America and Oceania. Advances in high-throughput sequencing and molecular testing provide powerful tools for molecular monitoring of artemisinin resistance in P. falciparum. This review summarizes the advances in molecular surveillance of artemisinin resistance in P. falciparum, aiming to provide insights into surveillance and management of artemisinin-resistant P. falciparum.

Key words: Plasmodium falciparum, Artemisinin resistance, Molecular surveillance, pfk13

CLC Number:

R531.3

LI Fei, LIU Yaobao, CAO Jun. Researches progress on molecular surveillance of artemisinin resistance in Plasmodium falciparum[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2025, 43(2): 260-268.

Figures/Tables 5

年份	调查地	样本量/份	pfk13突变频率/%	SNP突变频率/%				参考文献
年份	调查地	样本量/份	pfk13突变频率/%	C469Y	R561H	A675V	R622I	参考文献
2010	卢旺达胡耶	75	0	0	0	0	0	[57]
2014	卢旺达胡耶	81	2.5	0	0	0	0	[57]
2015	卢旺达胡耶	66	4.5	0	0	1.5	0	[57]
2019	卢旺达胡耶	66	12.1	0	4.5	1.5	0	[58]
2023	卢旺达胡耶	212	19.3	0	9.0	5.7	0	[59]
2013	卢旺达马萨卡和鲁胡哈	32	12.5	0	0	0	0	[60]
2014	卢旺达马萨卡和鲁胡哈	102	17.6	0	6.9	0	0	[60]
2015	卢旺达马萨卡和鲁胡哈	400	11.5	0.25	3.0	0	0	[60]
2014—2015	卢旺达马萨卡	257	-	0	7.4	0	0	[60]
2018	卢旺达马萨卡	51	21.6	0	19.6	0	0	[61]
2018	卢旺达鲁卡拉	82	28.1	0	21.9	0	0	[61]
2021	卢旺达鲁卡拉	135	31.9	0	23.5	6.4	0	[61]
2018	卢旺达	219	-	-	11.9	-	-	[5]
2018—2019	卢旺达基加利	73	30.1	0	21.9	0	0	[62]
1999—2004	乌干达	683	2.3	0	0	0	0	[63]
2012—2016	乌干达	716	3.6	0.14	0	0.14	0	[63]
2015	乌干达北部（古卢）	505	3.9	0	0	0	0	[65]
2017	乌干达北部（古卢）	87	9.2	0	0	9.2	0	[65]
2018	乌干达北部（古卢）	60	18.3	1.7	0	13.3	0	[65]
2019	乌干达北部（古卢）	93	20.4	4.3	0	11.8	0	[65]
2016	乌干达	326	4.6	1.2	0	0.6	0	[64]
2017	乌干达	419	4.8	2.1	0	2.4	0	[64]
2018	乌干达	500	5.6	2.6	0	1.4	0	[64]
2019	乌干达	762	7.6	2.7	0	4.3	0	[64]
2020	乌干达	1 181	17.8	9.4	0	6.5	0	[64]
2021	乌干达	933	17.3	4.8	1.2	5.9	0	[64]
2022	乌干达	940	27.6	9.6	1.4	10.1	0	[64]
2016	厄立特里亚	280	9.7	0	0.3	0	8.6	[66]
2017	厄立特里亚	211	8.6	0	0	0	7.6	[66]
2018	厄立特里亚	-	-	-	-	-	16.7	[5]
2019	厄立特里亚	361	22.8	0	0	0	21.1	[66]
2014	埃塞俄比亚阿姆哈拉	125	2.4	0	0	0	2.4	[67]
2018	埃塞俄比亚阿姆哈拉	598	-	-	-	-	9.8	[39]
2012—2015	赤道几内亚	224	6.7	0	0	0	0	[69]
2013	赤道几内亚	1	-	-	-	-	-	[69]
2016	坦桑尼亚	320	5.3	0	0	0	0	[70]
2017	坦桑尼亚	774	-	0	0.2	0	0	[71]
2019	坦桑尼亚	422	7.3	0	0.2	0	0	[72]
2020	坦桑尼亚	96	4.2	0	0	0	1.1	[73]
2022	坦桑尼亚	173	-	-	22.5	-	-	[74]
2010—2015	刚果（金）	66	0	0	0	0	0	[75]
2017	刚果（金）	717	1.0	0	0	0	0	[76]
2018—2019	刚果（金）	325	2.2	0	0	0	0	[77]
2020—2021	刚果（金）	1 065	3.2	0	0.094	0	0	[78]

References 88

[1]	World Health Organization. World malaria report 2023[R]. Geneva: World Health Organization, 2023: 7-8.
[2]	Noedl H, Se Y, Schaecher K, et al. Evidence of artemisinin-resistant malaria in western Cambodia[J]. N Engl J Med, 2008, 359(24): 2619-2620.
[3]	许秋利, 周鸿让, 钱门宝, 等. 疟疾经济负担研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(6): 749-752.
	Xu QL, Zhou HR, Qian MB, et al. Research progress on economic burden of malaria[J]. Chin J Parasitol Parasit Dis, 2020, 38(6): 749-752. (in Chinese)
[4]	张丽, 丰俊, 夏志贵, 等. 2019年全国疟疾疫情特征分析及消除工作进展[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(2): 133-138.
	Zhang L, Feng J, Xia ZG, et al. Epidemiological characteristics of malaria and progress on its elimination in China in 2019[J]. Chin J Parasitol Parasit Dis, 2020, 38(2): 133-138. (in Chinese)
[5]	Lu F, Culleton R, Zhang MH, et al. Emergence of indigenous artemisinin-resistant Plasmodium falciparum in Africa[J]. N Engl J Med, 2017, 376(10): 991-993.
[6]	WWARN K13 Genotype-Phenotype Study Group. Association of mutations in the Plasmodium falciparum Kelch13 gene (Pf3D7_1343700) with parasite clearance rates after artemisinin-based treatments-a WWARN individual patient data meta-analysis[J]. BMC Med, 2019, 17(1): 1.
[7]	WHO. Strategy to respond to antimalarial drug resistance in Africa[M]. Geneva: World Health Organization, 2022: 15-42.
[8]	World Health Organization. Report on antimalarial drug efficacy, resistance and response:10 years of surveillance (2010-2019)[M]. Geneva: World Health Organization, 2020: 9-17.
[9]	Ariey F, Witkowski B, Amaratunga C, et al. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria[J]. Nature, 2014, 505(7481): 50-55.
[10]	Straimer J, Gn?dig NF, Witkowski B, et al. Drug resistance. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates[J]. Science, 2015, 347(6220): 428-431.
[11]	Mbengue A, Bhattacharjee S, Pandharkar T, et al. A molecular mechanism of artemisinin resistance in Plasmodium falciparum malaria[J]. Nature, 2015, 520(7549): 683-687.
[12]	World Health Organization. Malaria: Artemisinin partial resistance[EB/OL]. (2025-01-09)[2025-03-18]. https://www.who.int/news-room/questions-and-answers/item/artemisinin-resistance.
[13]	Hunt P, Afonso A, Creasey A, et al. Gene encoding a deubiquitinating enzyme is mutated in artesunate- and chloroquine-resistant rodent malaria parasites[J]. Mol Microbiol, 2007, 65(1): 27-40.
[14]	Traub LM. Tickets to ride: Selecting cargo for clathrin-regulated internalization[J]. Nat Rev Mol Cell Biol, 2009, 10(9): 583-596.
[15]	Henriques G, Martinelli A, Rodrigues L, et al. Artemisinin resistance in rodent malaria: Mutation in the AP2 adaptor μ-chain suggests involvement of endocytosis and membrane protein trafficking[J]. Malar J, 2013, 12: 118.
[16]	Borrmann S, Straimer J, Mwai L, et al. Genome-wide screen identifies new candidate genes associated with artemisinin susceptibility in Plasmodium falciparum in Kenya[J]. Sci Rep, 2013, 3: 3318.
[17]	Birnbaum J, Scharf S, Schmidt S, et al. A Kelch13-defined endocytosis pathway mediates artemisinin resistance in malaria parasites[J]. Science, 2020, 367(6473): 51-59.
[18]	Henrici RC, van Schalkwyk DA, Sutherland CJ. Modification of pfap2μ and pfubp1 markedly reduces ring-stage susceptibility of Plasmodium falciparum to artemisinin in vitro[J]. Antimicrob Agents Chemother, 2019, 64(1): e01542-19.
[19]	Henriques G, Hallett RL, Beshir KB, et al. Directional selection at the pfmdr1, pfcrt, pfubp1, and pfap2mu loci of Plasmodium falciparum in Kenyan children treated with ACT[J]. J Infect Dis, 2014, 210(12): 2001-2008.
[20]	Adams T, Ennuson NAA, Quashie NB, et al. Prevalence of Plasmodium falciparum delayed clearance associated polymorphisms in adaptor protein complex 2 mu subunit (pfap2mu) and ubiquitin specific protease 1 (pfubp1) genes in Ghanaian isolates[J]. Parasit Vectors, 2018, 11(1): 175.
[21]	Amambua-Ngwa A, Amenga-Etego L, Kamau E, et al. Major subpopulations of Plasmodium falciparum in sub-Saharan Africa[J]. Science, 2019, 365(6455): 813-816.
[22]	章太婵, 梁雪雁, 韦华贵, 等. 2018—2020年赤道几内亚Bioko岛恶性疟原虫青蒿素耐药相关基因Pfubp1和Pfap2mu的单核苷酸多态性分析[J]. 中国血吸虫病防治杂志, 2023, 35(6): 557-564, 572.
	Zhang TC, Liang XY, Wei HG, et al. Single-nucleotide polymorphisms of artemisinin resistance-related Pfubp1 and Pfap2mu genes in Bioko Island, Equatorial Guinea from 2018 to 2020[J]. Chin J Schisto Control, 2023, 35(6): 557-564, 572. (in Chinese)
[23]	Sutherland CJ, Lansdell P, Sanders M, et al. pfk13-independent treatment failure in four imported cases of Plasmodium falciparum malaria treated with artemether-lumefantrine in the United Kingdom[J]. Antimicrob Agents Chemother, 2017, 61(3): e02382-16.
[24]	Olshina MA, Angrisano F, Marapana DS, et al. Plasmodium falciparum coronin organizes arrays of parallel actin filaments potentially guiding directional motility in invasive malaria parasites[J]. Malar J, 2015, 14: 280.
[25]	Demas AR, Sharma AI, Wong W, et al. Mutations in Plasmodium falciparum actin-binding protein coronin confer reduced artemisinin susceptibility[J]. Proc Natl Acad Sci USA, 2018, 115(50): 12799-12804.
[26]	Sharma AI, Shin SH, Bopp S, et al. Genetic background and PfKelch13 affect artemisinin susceptibility of PfCoronin mutants in Plasmodium falciparum[J]. PLoS Genet, 2020, 16(12): e1009266.
[27]	Otienoburu SD, Ma?ga-Ascofaré O, Schramm B, et al. Selection of Plasmodium falciparum pfcrt and pfmdr1 polymorphisms after treatment with artesunate-amodiaquine fixed dose combination or artemether-lumefantrine in Liberia[J]. Malar J, 2016, 15(1): 452.
[28]	Zhu HY, Zhu DQ, Wu K, et al. Establishment and evaluation of a qPCR method for the detection of pfmdr1 mutations in Plasmodium falciparum, the causal agent of fatal malaria[J]. Diagn Microbiol Infect Dis, 2024, 110(1): 116400.
[29]	Brown N, da Silva C, Webb C, et al. Antimalarial resistance risk in Mozambique detected by a novel quadruplex droplet digital PCR assay[J]. Antimicrob Agents Chemother, 2024, 68(7): e0034624.
[30]	Jiang TT, Huang Y, Cheng WJ, et al. Multiple single-nucleotide polymorphism detection for antimalarial pyrimethamine resistance via allele-specific PCR coupled with gold nanoparticle-based lateral flow biosensor[J]. Antimicrob Agents Chemother, 2021, 65(3): e01063-20.
[31]	Chahar M, Mishra N, Anvikar A, et al. Establishment and application of a novel isothermal amplification assay for rapid detection of chloroquine resistance (K76T) in Plasmodium falciparum[J]. Sci Rep, 2017, 7: 41119.
[32]	Khammanee T, Sawangjaroen N, Buncherd H, et al. A LAMP-SNP assay detecting C580Y mutation in Pfkelch13 gene from clinically dried blood spot samples[J]. Korean J Parasitol, 2021, 59(1): 15-22.
[33]	Zhu HY, Zhu DQ, Li YT, et al. Rapid detection of mutations in the suspected piperaquine resistance gene E415G-exo in Plasmodium falciparum exonuclease via AS-PCR and RAA with CRISPR/Cas12a[J]. Int J Parasitol Drugs Drug Resist, 2024, 26: 100568.
[34]	Bier FF, von Nickisch-Rosenegk M, Ehrentreich-F?rster E, et al. DNA microarrays[J]. Adv Biochem Eng Biotechnol, 2008, 109: 433-453.
[35]	Crameri A, Marfurt J, Mugittu K, et al. Rapid microarray-based method for monitoring of all currently known single-nucleotide polymorphisms associated with parasite resistance to antimalaria drugs[J]. J Clin Microbiol, 2007, 45(11): 3685-3691.
[36]	Maiga H, Morrison RD, Duffy PE. Sanger sequencing and deconvolution of polyclonal infections: A quantitative approach to monitor drug-resistant Plasmodium falciparum[J]. EBio Medicine, 2024, 103: 105115.
[37]	Nsanzabana C, Djalle D, Guérin PJ, et al. Tools for surveillance of anti-malarial drug resistance: An assessment of the current landscape[J]. Malar J, 2018, 17(1): 75.
[38]	Rogier E, Herman C, Huber CS, et al. Nationwide monitoring for Plasmodium falciparum drug-resistance alleles to chloroquine, sulfadoxine, and pyrimethamine, Haiti, 2016-2017[J]. Emerg Infect Dis, 2020, 26(5): 902-909.
[39]	Fola AA, Feleke SM, Mohammed H, et al. Plasmodium falciparum resistant to artemisinin and diagnostics have emerged in Ethiopia[J]. Nat Microbiol, 2023, 8(10): 1911-1919.
[40]	Holzschuh A, Lerch A, Gerlovina I, et al. Multiplexed ddPCR-amplicon sequencing reveals isolated Plasmodium falciparum populations amenable to local elimination in Zanzibar, Tanzania[J]. Nat Commun, 2023, 14(1): 3699.
[41]	Girgis ST, Adika E, Nenyewodey FE, et al. Drug resistance and vaccine target surveillance of Plasmodium falciparum using nanopore sequencing in Ghana[J]. Nat Microbiol, 2023, 8(12): 2365-2377.
[42]	de Cesare M, Mwenda M, Jeffreys AE, et al. Flexible and cost-effective genomic surveillance of P. falciparum malaria with targeted nanopore sequencing[J]. Nat Commun, 2024, 15(1): 1413.
[43]	Hassett MR, Roepe PD. Origin and spread of evolving artemisinin-resistant Plasmodium falciparum malarial parasites in Southeast Asia[J]. Am J Trop Med Hyg, 2019, 101(6): 1204-1211.
[44]	Rovira-Vallbona E, Kattenberg JH, Hong NV, et al. Molecular surveillance of Plasmodium falciparum drug-resistance markers in Vietnam using multiplex amplicon sequencing (2000-2016)[J]. Sci Rep, 2023, 13(1): 13948.
[45]	Quang Bui P, Hong Huynh Q, Thanh Tran D, et al. Pyronaridine-artesunate efficacy and safety in uncomplicated Plasmodium falciparum malaria in areas of artemisinin-resistant falciparum in Viet Nam (2017-2018)[J]. Clin Infect Dis, 2020, 70(10): 2187-2195.
[46]	Quang HH, Chavchich M, Trinh NTM, et al. Multidrug-resistant Plasmodium falciparum parasites in the central Highlands of Vietnam jeopardize malaria control and elimination strategies[J]. Antimicrob Agents Chemother, 2021, 65(4): e01639-20.
[47]	Imwong M, Hien TT, Thuy-Nhien NT, et al. Spread of a single multidrug resistant malaria parasite lineage (PfPailin) to Vietnam[J]. Lancet Infect Dis, 2017, 17(10): 1022-1023.
[48]	Imwong M, Suwannasin K, Kunasol C, et al. The spread of artemisinin-resistant Plasmodium falciparum in the Greater Mekong subregion: A molecular epidemiology observational study[J]. Lancet Infect Dis, 2017, 17(5): 491-497.
[49]	Chaisatit C, Sai-Ngam P, Thaloengsok S, et al. Molecular detection of mutations in the propeller domain of kelch 13 and pfmdr1 copy number variation in Plasmodium falciparum isolates from Thailand collected from 2002 to 2007[J]. Am J Trop Med Hyg, 2021, 105(4): 1093-1096.
[50]	Boonyalai N, Thamnurak C, Sai-Ngam P, et al. Plasmodium falciparum phenotypic and genotypic resistance profile during the emergence of piperaquine resistance in Northeastern Thailand[J]. Sci Rep, 2021, 11(1): 13419.
[51]	Khammanee T, Sawangjaroen N, Buncherd H, et al. Molecular surveillance of Pfkelch13 and Pfmdr1 mutations in Plasmodium falciparum isolates from southern Thailand[J]. Korean J Parasitol, 2019, 57(4): 369-377.
[52]	Kobasa T, Talundzic E, Sug-Aram R, et al. Emergence and spread of kelch13 mutations associated with artemisinin resistance in Plasmodium falciparum parasites in 12 Thai Provinces from 2007 to 2016[J]. Antimicrob Agents Chemother, 2018, 62(4): e02141-17.
[53]	Anderson TJC, Nair S, McDew-White M, et al. Population parameters underlying an ongoing soft sweep in Southeast Asian malaria parasites[J]. Mol Biol Evol, 2017, 34(1): 131-144.
[54]	Kagoro FM, Barnes KI, Marsh K, et al. Mapping genetic markers of artemisinin resistance in Plasmodium falciparum malaria in Asia: A systematic review and spatiotemporal analysis[J]. Lancet Microbe, 2022, 3(3): e184-e192.
[55]	Dwivedi A, Reynes C, Kuehn A, et al. Functional analysis of Plasmodium falciparum subpopulations associated with artemisinin resistance in Cambodia[J]. Malar J, 2017, 16(1): 493.
[56]	World Health Organization Regional Office for the Eastern Mediterranean. Country cooperation strategy for WHO and Somalia 2021-2025[M]. Cairo: World Health Organization Regional Office for the Eastern Mediterranean, 2022: 5-10.
[57]	Tacoli C, Gai PP, Bayingana C, et al. Artemisinin resistance-associated K13 polymorphisms of Plasmodium falciparum in southern Rwanda, 2010-2015[J]. Am J Trop Med Hyg, 2016, 95(5): 1090-1093.
[58]	Bergmann C, van Loon W, Habarugira F, et al. Increase in kelch 13 polymorphisms in Plasmodium falciparum, southern Rwanda[J]. Emerg Infect Dis, 2021, 27(1): 294-296.
[59]	van Loon W, Schallenberg E, Igiraneza C, et al. Escalating Plasmodium falciparum K13 marker prevalence indicative of artemisinin resistance in southern Rwanda[J]. Antimicrob Agents Chemother, 2024, 68(1): e0129923.
[60]	Uwimana A, Legrand E, Stokes BH, et al. Emergence and clonal expansion of in vitro artemisinin-resistant Plasmodium falciparum kelch13 R561H mutant parasites in Rwanda[J]. Nat Med, 2020, 26(10): 1602-1608.
[61]	Uwimana A, Umulisa N, Venkatesan M, et al. Association of Plasmodium falciparum kelch13 R561H genotypes with delayed parasite clearance in Rwanda: An open-label, single-arm, multicentre, therapeutic efficacy study[J]. Lancet Infect Dis, 2021, 21(8): 1120-1128.
[62]	Straimer J, Gandhi P, Renner KC, et al. High prevalence of Plasmodium falciparum K13 mutations in Rwanda is associated with slow parasite clearance after treatment with artemether-lumefantrine[J]. J Infect Dis, 2022, 225(8): 1411-1414.
[63]	Conrad MD, Nsobya SL, Rosenthal PJ. The diversity of the Plasmodium falciparum K13 propeller domain did not increase after implementation of artemisinin-based combination therapy in Uganda[J]. Antimicrob Agents Chemother, 2019, 63(10): e01234-19.
[64]	Conrad MD, Asua V, Garg S, et al. Evolution of partial resistance to artemisinins in malaria parasites in Uganda[J]. N Engl J Med, 2023, 389(8): 722-732.
[65]	Balikagala B, Fukuda N, Ikeda M, et al. Evidence of artemisinin-resistant malaria in Africa[J]. N Engl J Med, 2021, 385(13): 1163-1171.
[66]	Mihreteab S, Platon L, Berhane A, et al. Increasing prevalence of artemisinin-resistant HRP2-negative malaria in Eritrea[J]. N Engl J Med, 2023, 389(13): 1191-1202.
[67]	Bayih AG, Getnet G, Alemu A, et al. A unique Plasmodium falciparum K13 gene mutation in northwest Ethiopia[J]. Am J Trop Med Hyg, 2016, 94(1): 132-135.
[68]	Xu WJ, Zhang X, Chen HL, et al. Molecular markers associated with drug resistance in Plasmodium falciparum parasites in central Africa between 2016 and 2021[J]. Front Public Health, 2023, 11: 1239274.
[69]	Huang F, Yan H, Xue JB, et al. Molecular surveillance of pfcrt, pfmdr1 and pfk13-propeller mutations in Plasmodium falciparum isolates imported from Africa to China[J]. Malar J, 2021, 20(1): 73.
[70]	Ishengoma DS, Mandara CI, Francis F, et al. Efficacy and safety of artemether-lumefantrine for the treatment of uncomplicated malaria and prevalence of Pfk13 and Pfmdr1 polymorphisms after a decade of using artemisinin-based combination therapy in mainland Tanzania[J]. Malar J, 2019, 18(1): 88.
[71]	Moser KA, Madebe RA, Aydemir O, et al. Describing the current status of Plasmodium falciparum population structure and drug resistance within mainland Tanzania using molecular inversion probes[J]. Mol Ecol, 2021, 30(1): 100-113.
[72]	Bwire GM, Ngasala B, Mikomangwa WP, et al. Detection of mutations associated with artemisinin resistance at k13-propeller gene and a near complete return of chloroquine susceptible falciparum malaria in Southeast of Tanzania[J]. Sci Rep, 2020, 10(1): 3500.
[73]	Bakari C, Mandara CI, Madebe RA, et al. Trends of Plasmodium falciparum molecular markers associated with resistance to artemisinins and reduced susceptibility to lumefantrine in Mainland Tanzania from 2016 to 2021[J]. Malar J, 2024, 23(1): 71.
[74]	Ishengoma DS, Mandara CI, Bakari C, et al. Evidence of artemisinin partial resistance in northwestern Tanzania: Clinical and molecular markers of resistance[J]. Lancet Infect Dis, 2024, 24(11): 1225-1233.
[75]	Voumbo-Matoumona DF, Akiana J, Madamet M, et al. High prevalence of Plasmodium falciparum antimalarial drug resistance markers in isolates from asymptomatic patients from the Republic of the Congo between 2010 and 2015[J]. J Glob Antimicrob Resist, 2018, 14: 277-283.
[76]	Yobi DM, Kayiba NK, Mvumbi DM, et al. The lack of K13-propeller mutations associated with artemisinin resistance in Plasmodium falciparum in Democratic Republic of Congo (DRC)[J]. PLoS One, 2020, 15(8): e0237791.
[77]	Yobi DM, Kayiba NK, Mvumbi DM, et al. Assessment of Plasmodium falciparum anti-malarial drug resistance markers in pfk13-propeller, pfcrt and pfmdr1 genes in isolates from treatment failure patients in Democratic Republic of Congo, 2018-2019[J]. Malar J, 2021, 20(1): 144.
[78]	Mesia Kahunu G, Wellmann Thomsen S, Wellmann Thomsen L, et al. Identification of the PfK13 mutations R561H and P441L in the democratic republic of Congo[J]. Int J Infect Dis, 2024, 139: 41-49.
[79]	Chenet SM, Akinyi Okoth S, Huber CS, et al. Independent emergence of the Plasmodium falciparum Kelch propeller domain mutant allele C580Y in Guyana[J]. J Infect Dis, 2016, 213(9): 1472-1475.
[80]	Mathieu LC, Cox H, Early AM, et al. Local emergence in Amazonia of Plasmodium falciparum k13 C580Y mutants associated with in vitro artemisinin resistance[J]. eLife, 2020, 9: e51015.
[81]	Vanhove M, Schwabl P, Clementson C, et al. Temporal and spatial dynamics of Plasmodium falciparum clonal lineages in Guyana[J]. bioRxiv, 2024: 2024.01.31.578156.
[82]	Stokes BH, Dhingra SK, Rubiano K, et al. Plasmodium falciparum K13 mutations in Africa and Asia impact artemisinin resistance and parasite fitness[J]. eLife, 2021, 10: e66277.
[83]	Lucchi NW, Abdallah R, Louzada J, et al. Molecular surveillance for polymorphisms associated with artemisinin-based combination therapy resistance in Plasmodium falciparum isolates collected in the state of Roraima, Brazil[J]. Am J Trop Med Hyg, 2020, 102(2): 310-312.
[84]	Miotto O, Sekihara M, Tachibana SI, et al. Emergence of artemisinin-resistant Plasmodium falciparum with kelch13 C580Y mutations on the island of New Guinea[J]. PLoS Pathog, 2020, 16(12): e1009133.
[85]	Yoshida N, Yamauchi M, Morikawa R, et al. Increase in the proportion of Plasmodium falciparum with kelch13 C580Y mutation and decline in pfcrt and pfmdr1 mutant alleles in Papua New Guinea[J]. Malar J, 2021, 20(1): 410.
[86]	Prosser C, Meyer W, Ellis J, et al. Resistance screening and trend analysis of imported falciparum malaria in NSW, Australia (2010 to 2016)[J]. PLoS One, 2018, 13(5): e0197369.
[87]	Ménard D, Khim N, Beghain J, et al. A worldwide map of Plasmodium falciparum K13-propeller polymorphisms[J]. N Engl J Med, 2016, 374(25): 2453-2464.
[88]	张苍林, 聂仁华, 徐丹, 等. 中缅边境地区恶性疟原虫Pfcrt、Pfmdr和PfK13基因多态性与体外药物敏感性相关性的分析[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(5): 580-588.
	Zhang CL, Nie RH, Xu D, et al. Correlation of Pfcrt, Pfmdr and PfK13 gene polymorphisms and in vitro drug susceptibility of Plasmodium falciparum isolates from China-Myanmar border region[J]. Chin J Parasitol Parasit Dis, 2020, 38(5): 580-588. (in Chinese)

方法	优点	缺点
PCR + RFLP	技术成熟，操作简便	灵敏度和特异性相对较低，难以检测低丰度突变
qPCR	特异性强，灵敏度高，结果快速准确	需要昂贵的荧光定量设备
ddPCR	高灵敏度，高特异性，可以对低丰度目标进行定量	成本较高，设备和技术要求严格
等温扩增技术	简便快速，不需要复杂设备，适合现场检测	难以对多个靶标进行扩增，限制了其在多个耐药基因检测中的应用
微阵列芯片	可以同时检测多个耐药基因SNP，高通量	定制化芯片成本高，技术要求高
一代测序（Sanger测序）	读长较长，准确度高，金标准	测序通量低，成本较高
二代测序	高通量，可同时测序大量DNA片段，效率高	数据处理复杂，成本较高
三代测序	长读长，适用于复杂基因组分析	成本高，技术相对较新，需要进一步优化