Prevalence of K13-propeller polymorphisms in Plasmodium falciparum from China-Myanmar border in 2007–2012
© Wang et al.; licensee BioMed Central. 2015
Received: 23 January 2015
Accepted: 16 March 2015
Published: 18 April 2015
The recent emergence and spread of artemisinin resistance in the Greater Mekong Subregion poses a great threat to malaria control and elimination. A K13-propeller gene (K13), PF3D7_1343700, has been associated lately with artemisinin resistance both in vitro and in vivo. This study aimed to investigate the K13 polymorphisms in Plasmodium falciparum parasites from the China-Myanmar border area where artemisinin use has the longest history.
A total of 180 archived P. falciparum isolates containing 191 parasite clones, mainly collected in 2007–2012 from the China-Myanmar area, were used to obtain the full-length K13 gene sequences.
Seventeen point mutations were identified in 46.1% (88/191) parasite clones, of which seven were new. The F446I mutation predominated in 27.2% of the parasite clones. The C580Y mutation that is correlated with artemisinin resistance was detected at a low frequency of 1.6%. Collectively, 43.1% of the parasite clones contained point mutations in the kelch domain of the K13 gene. Moreover, there was a trend of increase in the frequency of parasites carrying kelch domain mutations through the years of sample collection. In addition, a microsatellite variation in the N-terminus of the K13 protein was found to have reached a high frequency (69.1%).
This study documented the presence of mutations in the K13 gene in parasite populations from the China-Myanmar border. Mutations present in the kelch domain have become prevalent (>40%). A predominant mutation F446I and a prevalent microsatellite variation in the N-terminus were identified, but their importance in artemisinin resistance remains to be elucidated.
KeywordsMalaria Plasmodium falciparum artemisinin resistance K13 gene F3D7_1343700
Malaria has been scourging human beings for millennia, and remains responsible for over 430,000 child deaths in Africa every year . The world has made remarkable strides in battling against this ancient enemy during the past decade, reducing by an impressive 47% in mortality rate globally between 2000 and 2013 . However, parasite resistance to anti-malarials remains an ever-present obstacle to eliminate malaria. Chloroquine and sulphadoxine-pyrimethamine have failed as crucial medicines in the treatment of the deadly malaria parasite Plasmodium falciparum due to the emergence and rapid spread of drug resistance. More worryingly, resistance to artemisinin (ART) family drugs has been detected and is spreading in Southeast Asia [2-4], posing a major threat to the implementation of artemisinin-based combination therapy (ACT) as a defensive line against P. falciparum.
ART resistance is manifested clinically as delayed parasite clearance half-life (>5 hours) in vivo [4,5]. An in vitro ring-stage survival assay (RSA0-3h), which measures the percentage of early ring-stage parasites (0–3 hrs post-invasion of red blood cells (RBCs)) that survive exposure to a pharmacologically relevant concentration of dihydroartemisinin, has been developed to reflect this ART resistance phenotype . Recent work has associated ART resistance with mutations in the propeller domain of a kelch gene on chromosome 13 (PF3D7_1343700, K13 gene) . The K13 mutation M476I was initially identified in a Tanzanian P. falciparum strain that had undergone in vitro ART selection for five years. Research on parasite isolates from Cambodia, where ART resistance was first observed, identified K13 mutations Y493H, R539T and C580Y to be associated with delayed clearance . These mutations were confirmed to contribute to in vitro ART resistance through genetic manipulations of the K13 gene [7,8]. A large, multicentre, clinical study further indicates that ART resistance is spreading in the Greater Mekong Subregion (GMS), where single-point mutations in the propeller domain of K13 after the position 440 are collectively associated with ART resistance . Surveys conducted in different regions showed that K13 mutations associated with ART resistance were restricted to certain areas of the GMS, including Cambodia, Thailand, Myanmar, and Vietnam. The C580Y mutation is the predominant one approaching fixation in Western Cambodia [5,9-11]. These mutations have not been detected in Bangladesh and Laos [4,10,12]. Surveys of African parasite populations, while having found a diverse array of mutations within the K13 gene, did not detect those mutations associated with ART resistance [13-17].
ART family drugs have been used in China’s Yunnan Province since the late 1970s . In recent years, clinical efficacy studies conducted in this region showed that artemisinin drugs for treating falciparum malaria remain highly effective [19,20]. However, the proportion of day 3 parasite-positive cases in one study reached 18.5% , well above the 10% threshold set by the World Health Organization as a proxy indicator of suspected ART resistance , suggesting possible emergence of ART resistance in this area. In this study, the polymorphisms of K13 genes in parasite populations along the China-Myanmar border were investigated, and the presence of K13 mutations that are associated with clinical ART resistance in this region was demonstrated using longitudinally archived parasite samples. More importantly, parasite strains carrying wild-type K13 alleles have been declining through the six years of sample collection.
Collection of parasite clinical isolates, DNA extraction and genotyping
Plasmodium falciparum clinical isolates were collected during the period 2004–2012 in malaria clinics located along the China-Myanmar border, cultured and archived . A total of 180 samples were analysed including two collected in 2004, 25 in 2007, 47 in 2008, 78 in 2009, 11 in 2010, five in 2011, and 12 in 2012. Genomic DNA was extracted using the Wizard® Genomic DNA Purification Kit (Promega, WI, USA). Parasite samples were genotyped at msp1, msp2 and glurp using previously described methods [23-25] to distinguish single from mixed-strain infections .
K13 propeller gene amplification and sequencing analysis
The full-length K13 gene was amplified by high-fidelity PCR using Advantage HD DNA Polymerase Mix (Clontech, Mountain View, CA, USA) and primers KP13-F (5′-TATAACAAGGCGTAAATATTCGTG-3′) and KP13-R (5′-TGTGCATGAAAATAAATATTAAAGAAG-3′). PCR reactions were performed in 37.5 μl with 0.5 μM of DNA template, 0.2 μM of each primer, 3.75 μl of 10 × PCR buffer, 3.75 units of DNA polymerase mix, and 0.2 mM of dNTP mix. Reaction conditions consisted of an initial denaturation at 95°C for 5 min followed by 35 cycles of 95°C for 30 sec, 55°C for 30 sec and 68°C for 3 min, and a final extension step for 7 min at 68°C. PCR products were sequenced in both directions using sequencing primers KP13-65 F (5′-GGGAATCTGGTGGTAACAGC-3′), KP13-640R (5′-CACTAGCATCACTTAATTCCGTT-3′), KP13-517 F (5′-GATGCAGCAAATCTTATAAATGATG-3′), KP13-759 F (5′-GGAAAGAGTACGATTGTACAAAG-3′), KP13-1363R (5′-CTACACCATCAAATCCACCTATA-3′), KP13-1595 F (5′-GTGGTGTTACGTCAAATGGTAG-3′), and KP13-R. Sequences were assembled by DNASTAR (WI, USA) with manual editing. Alignment of DNA sequences were performed using MEGA 6.0  with the K13 sequence of the 3D7 clone (PF3D7_1343700) retrieved from PlasmoDB as the reference. To superimpose the mutations in the K13 protein, the 3D structure of the K13 protein was predicted by Phre2 online protein structure prediction tool . The chosen template retained 100% confidence based on homology assessment and model prediction quality.
Fisher’s exact test was done to assess difference in the frequency of mutations between years using GraphPad Prism 5 (GraphPad Software, Inc. La Jolla, CA, USA).
The full-length K13-propeller genes were sequenced from a total of 180 clinical isolates collected from malaria patients along the China-Myanmar border. Of these isolates, 178 were collected during 2007–2012, while two were collected in 2004. Genotyping of these parasites at three polymorphic genes confirmed that 169 were monoclonal infections, whereas 11 were mixed infections at one of the three loci . Since each of the mixed infections contained only two allelic types, they were considered as having two parasite strains in each sample. Based on their clear sequencing chromatograms, these mixed infections were also included in the analysis. Accordingly, the frequencies of mutations were estimated by using 191 parasite clones in the study population.
Amino acid substitutions in the K13 gene in parasites from the China-Myanmar border (n = 191)
In kelch domain
The involvement of K13 mutations in ART resistance in Cambodia encouraged molecular surveillance of K13 genes in many malaria-endemic regions. K13 mutations associated with ART resistance were mainly detected in areas of the GMS [5,9-11], with C580Y being the predominant. In contrast, surveys in Africa identified numerous mutations in the K13 gene, but most occurred at low frequencies. Moreover, mutations associated with ART resistance were not observed [13-16]. In this study, sequencing of K13 gene from 180 longitudinally collected parasite samples (containing 191 parasite clones) from the China-Myanmar border area identified 17 mutations (ten previously described and seven new). Compared with other surveys in Myanmar or the China-Myanmar border, parasites from this study shared three mutations (F446I, P574L and A676D) with those reported by Feng et al. , three (P441L, N458Y and C580Y) with those reported by Nyunt et al.  and four (F446I, P574L, C580Yand A676D) with those reported by Tun et al. . Of particular interest is the identification of the F446I as the predominant mutation in the study samples, which has reached a frequency of 27.1%, coincident with results from other study (19.2%) in this region . Although other mutations were at low frequencies, they collectively gave a total of 41.3% parasites carrying mutations in the kelch domain. Among them, the predominant C580Y mutation in other regions of Southeast Asia [4,5,10] was detected in 1.6% of these samples. This mutation was found to be significantly associated with prolonged parasite clearance half-life in parasites from Cambodia  and was confirmed to confer ART resistance by genetic manipulations [7,8]. Most of the mutations in the kelch domain, including the most prevalent F446I mutation, remain to be genetically characterized to determine whether they confer ART resistance.
Another intriguing phenomenon discovered in this study is the highly prevalent microsatellite variations in the N-terminus of the K13 gene. Compared to the wild-type parasite, which has six N residues, 69.1% parasites harboured eight N residues. The eight N variation was only reported in Senegalese isolates with a frequency of 6.3% . More interestingly, the predominant mutation F446I was observed only in parasites with eight N repeats. In an earlier study, microsatellite variations in the nhe1 gene were associated with altered sensitivities to quinine in parasite population from the same area . Therefore, it would be interesting to find out whether the K13 microsatellite variations affect parasites’ sensitivities to ART drugs.
ARTs have been used in the China-Myanmar border area for over three decades, mostly as monotherapy prior to 2005. The ACT drug deployed here is the dihydroartemisinin/piperaquine combination, compared to artesunate/mefloquine in most other areas of the GMS. One can hypothesize that it is likely that the distinct K13 mutations observed in parasites from this study site might have emerged separately as a result of selection from a different ART regimen. In addition, given the predominant status of the C580Y mutation in other parts of the GMS, it remains an open question as to whether the presence of this mutation in these samples was due to independent emergence or had spread from other areas. Furthermore, clinical efficacy studies also detected day-3 parasite positivity rate to >10% after artesunate treatment of falciparum malaria in this area . Taken together, increased surveillance and population studies are needed to determine further spread of ART resistance in the GMS.
Seventeen point mutations were identified in the K13 gene from 180 archived parasite samples collected from the China-Myanmar border area. Analysis of the 191 parasite strains identified 43.1% carrying point mutations in the kelch domain, of which the F446I mutation reached a predominance of 27.1%. Given the presence of K13 mutations conferring ART resistance and detection of day-3 parasite positivity after ART drug treatment, increased surveillance of P. falciparum for anti-malarial drug resistance is highly demanded.
This work was supported by grant from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (U19 AI089672).
- WHO. World malaria report 2014. Geneva: World Health Organization; 2014. http://www.who.int/malaria/publications/world_malaria_report_2014/en/ 2014.Google Scholar
- Noedl H, Se Y, Schaecher K, Smith BL, Socheat D, Fukuda MM, et al. Evidence of artemisinin-resistant malaria in Western Cambodia. N Engl J Med. 2008;359:2619–20.View ArticlePubMedGoogle Scholar
- Dondorp AM, Nosten F, Yi P, Das D, Phyo AP, Tarning J, et al. Artemisinin resistance in Plasmodium falciparum malaria. N Engl J Med. 2009;361:455–67.View ArticlePubMed CentralPubMedGoogle Scholar
- 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;371:411–23.View ArticlePubMed CentralPubMedGoogle Scholar
- 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;505:50–5.View ArticlePubMedGoogle Scholar
- Witkowski B, Amaratunga C, Khim N, Sreng S, Chim P, Kim S, et al. Novel phenotypic assays for the detection of artemisinin-resistant Plasmodium falciparum malaria in Cambodia: in-vitro and ex-vivo drug-response studies. Lancet Infect Dis. 2013;13:1043–9.View ArticlePubMedGoogle Scholar
- Straimer JGN, Witkowski B, Amaratunga C, Duru V, Ramadani AP, Dacheux M, et al. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates. Science. 2015;347:428–31.View ArticlePubMedGoogle Scholar
- Ghorbal M, Gorman M, Macpherson CR, Martins RM, Scherf A, Lopez-Rubio JJ. Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system. Nat Biotechnol. 2014;32:819–21.View ArticlePubMedGoogle Scholar
- Nyunt MH, Hlaing T, Oo HW, Tin-Oo LK, Phway HP, Wang B, et al. Molecular assessment of artemisinin-resistance markers, polymorphisms in the K13 propeller and a multidrug-resistance gene, in eastern and western border areas of Myanmar. Clin Infect Dis. 2014, doi:10.1093/cid/ciu1160.
- Takala-Harrison S, Jacob CG, Arze C, Cummings MP, Silva JC, Dondorp AM, et al. Independent emergence of artemisinin resistance mutations among Plasmodium falciparum in Southeast Asia. J Infect Dis. 2014, doi:10.1093/infdis/jiu491.
- Thriemer K, Hong N, Rosanas-Urgell A, Phuc BQ, Ha-do M, Pockele E. Delayed parasite clearance after treatment with dihydroartemisinin-piperaquine in Plasmodium falciparum malaria patients in Central Vietnam. Antimicrob Agents Chemother. 2014;58(12):7049–55.View ArticlePubMedGoogle Scholar
- Mohon A, Alam MS, Bayih AG, Folefoc A, Shahinas D, Haque R. Mutations in Plasmodium falciparum K13 propeller gene from Bangladesh (2009–2013). Malar J. 2014;13:431. doi:10.1186/1475-2875-13-431.View ArticlePubMed CentralPubMedGoogle Scholar
- Conrad MD, Bigira V, Kapisi J, Muhindo M, Kamya MR, Havlir DV, et al. Polymorphisms in K13 and falcipain-2 associated with artemisinin resistance are not prevalent in Plasmodium falciparum isolated from Ugandan children. Plos One. 2014;9:e105690.View ArticlePubMed CentralPubMedGoogle Scholar
- Taylor SM, Parobeck CM, DeConti DK, Kayentao K, Coulibaly SO, Greenwood BM, et al. Absence of putative artemisinin resistance mutations among Plasmodium falciparum in sub-Saharan Africa: a molecular epidemiologic study. J Infect Dis. 2014;211:680–8. doi: 10.1093/infdis/jiu467.View ArticlePubMedGoogle Scholar
- Kamau E, Campino S, Amenga-Etego L, Drury E, Ishengoma D, Johnson K, et al. K13-propeller polymorphisms in Plasmodium falciparum parasites from sub-Saharan Africa. J Infect Dis. 2014, doi: 10.1093/infdis/jiu608.
- Torrentino-Madamet M, Fall B, Benoit N, Camara C, Amalvict R, Fall M. Limited polymorphisms in k13 gene in Plasmodium falciparum isolates from Dakar, Senegal in 2012–2013. Malar J. 2014;13:472. doi:410.1186/1475-2875-1113-1472.View ArticlePubMed CentralPubMedGoogle Scholar
- Isozumi R, Uemura H, Kimata I, Ichinose Y, Logedi J, Omar AH, et al. Novel mutations in K13 propeller gene of artemisinin-resistant Plasmodium falciparum. Emerg Infect Dis. 2015;21:40–2. doi:10.3201/eid2103.140898.View ArticleGoogle Scholar
- Cui L, Su XZ. Discovery, mechanisms of action and combination therapy of artemisinin. Expert Rev Anti Infect Ther. 2009;7:999–1013.View ArticlePubMed CentralPubMedGoogle Scholar
- Sun X, Zhang Z, Wang J, Deng Y, Yang Y, Lasi J, et al. Therapeutic efficacy and safety of compound dihydroartemisinin/piperaquine for uncomplicated Plasmodium falciparum infection in Laiza city of Myanmar bordering on China. Chin J Parasitol Parasit Dis. 2011;29:372–5.Google Scholar
- Huang F, Tang L, Yang H, Zhou S, Sun X, Liu H. Therapeutic efficacy of artesunate in the treatment of uncomplicated Plasmodium falciparum malaria and anti-malarial, drug-resistance marker polymorphisms in populations near the China-Myanmar border. Malar J. 2012;11:278.View ArticlePubMed CentralPubMedGoogle Scholar
- WHO. Global plan for artemisinin resistance containment (GPARC). 2011. http://who.int/malaria/publications/atoz/9789241500838/en/.Google Scholar
- Wang Z, Parker D, Meng H, Wu L, Li J, Zhao Z, et al. In vitro sensitivity of Plasmodium falciparum from China-Myanmar border area to major ACT drugs and polymorphisms in potential target genes. PLoS One. 2012;7:e30927.View ArticlePubMed CentralPubMedGoogle Scholar
- Kaneko O, Kimura M, Kawamoto F, Ferreira MU, Tanabe K. Plasmodium falciparum: allelic variation in the merozoite surface protein 1 gene in wild isolates from southern Vietnam. Exp Parasitol. 1997;86:45–57.View ArticlePubMedGoogle Scholar
- Snounou G, Zhu X, Siripoon N, Jarra W, Thaithong S, Brown KN, et al. Biased distribution of msp1 and msp2 allelic variants in Plasmodium falciparum populations in Thailand. Trans R Soc Trop Med Hyg. 1999;93:369–74.View ArticlePubMedGoogle Scholar
- Roper C, Richardson W, Elhassan IM, Giha H, Hviid L, Satti GM, et al. Seasonal changes in the Plasmodium falciparum population in individuals and their relationship to clinical malaria: a longitudinal study in a Sudanese village. Parasitology. 1998;116:501–10.View ArticlePubMedGoogle Scholar
- Meng H, Zhang R, Yang H, Fan Q, Su X, Miao J, et al. In vitro sensitivity of Plasmodium falciparum clinical isolates from the China-Myanmar border area to quinine and association with polymorphism in the Na+/H+ exchanger. Antimicrob Agents Chemother. 2010;54:4306–13.View ArticlePubMed CentralPubMedGoogle Scholar
- Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30:2725–9.View ArticlePubMed CentralPubMedGoogle Scholar
- Kelley LA, Sternberg MJE. Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc. 2009;4:363–71.View ArticlePubMedGoogle Scholar
- Yuan LL, Zhao H, Wu LO, Li XM, Parker D, Xu SH, et al. Plasmodium falciparum populations from northeastern Myanmar display high levels of genetic diversity at multiple antigenic loci. Acta Trop. 2013;125:53–9.View ArticlePubMed CentralPubMedGoogle Scholar
- Feng J, Zhou D, Lin Y, Xiao H, Yan H, Xia Z. Amplification of pfmdr1, pfcrt, pvmdr1 and K13-propeller polymorphism associated with Plasmodium falciparum and Plasmodium vivax at the China-Myanmar border. Antimicrob Agents Chemother 2015, doi: 10.1128/AAC.04843-14
- Tun KM, Imwong M, Lwin KM, Win AA, Hlaing TM, Hlaing T, et al. Spread of artemisinin-resistant Plasmodium falciparum in Myanmar: a cross-sectional survey of the K13 molecular marker. Lancet Infect Dis. 2015, doi:10.1016/S1473-3099(15)70032-0.
- Huang F, Tang L, Yang H, Zhou S, Liu H, Li J, et al. Molecular epidemiology of drug resistance markers of Plasmodium falciparum in Yunnan Province, China. Malar J. 2012;11:243.View ArticlePubMed CentralPubMedGoogle Scholar
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