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Increase in the proportion of Plasmodium falciparum with kelch13 C580Y mutation and decline in pfcrt and pfmdr1 mutant alleles in Papua New Guinea

Abstract

Background

The C580Y mutation in the Plasmodium falciparum kelch13 gene is the most commonly observed variant in artemisinin-resistant isolates in the Greater Mekong Subregion (GMS). Until 2017, it had not been identified outside the GMS, except for Guyana/Amazonia. In 2017, three parasites carrying the C580Y mutation were identified in Papua New Guinea (PNG). As the C580Y allele rapidly spread in the GMS, there is concern that this mutant is now spreading in PNG.

Methods

In 2020, a cross-sectional survey was conducted at two clinics in Wewak, PNG. Symptomatic patients infected with P. falciparum were treated with artemether plus lumefantrine following a national treatment policy. Blood samples were obtained before treatment, and polymorphisms in kelch13, pfcrt, and pfmdr1 were determined. Parasite positivity was examined on day 3. The results were compared with those of previous studies conducted in 2002, 2003, and 2016–2018.

Results

A total of 94 patients were included in this analysis. The proportion of C580Y was significantly increased (2.2% in 2017, 5.7% in 2018, and 6.4% in 2020; p = 4.2 × 10–3). A significant upward trend was observed in the wild-type proportion for pfcrt (1.9% in 2016 to 46.7% in 2020; p = 8.9 × 10–16) and pfmdr1 (59.5% in 2016 to 91.4% in 2020; p = 2.3 × 10–6). Among 27 patients successfully followed on day 3, including three with C580Y infections, none showed positive parasitaemia.

Conclusions

Under the conditions of significant increases in pfcrt K76 and pfmdr1 N86 alleles in PNG, the increase in kelch13 C580Y mutants may be a warning indicator of the emergence of parasites resistant to the currently used first-line treatment regimen of artemether plus lumefantrine. Therefore, nationwide surveillance of molecular markers for drug resistance and assessment of its therapeutic effects are important.

Background

Artemisinin (ART)-based combination therapy (ACT) is a widely used first-line treatment for uncomplicated malaria. Malaria deaths have markedly decreased since the introduction of the treatment in the early 2000s [1]. However, the emergence of ART-resistant Plasmodium falciparum was first reported in the Greater Mekong Subregion (GMS) in 2006 [2]. Since then, ART-resistant parasites have rapidly spread in the region, partly because of the emergence of resistance to partner drug(s) of ACT [3, 4]. Therefore, the emergence and spread of ART-resistant parasites outside the GMS has become a global concern.

Propeller polymorphisms of the kelch13 are useful molecular markers for monitoring the emergence and spread of ART resistance [3, 5]. To date, ten non-synonymous mutations in kelch13 have been validated as polymorphisms for ART resistance. These include F446I, N458Y, M476I, Y493H, R539T, I543T, P553L, R561H, P574L, and C580Y [6]. In particular, C580Y has gradually outcompeted the other mutations and become dominant in some parts of the GMR region [7, 8]. C580Y is considered the most useful molecular marker for tracing the spread of ART resistance in GMS. However, outside the GMS region, this mutation has been detected only in Guyana/Amazonia [9, 10] and, more recently, in Papua New Guinea (PNG) [11].

In PNG, ACT was officially introduced as the first-line treatment regimen for uncomplicated malaria in 2010. Until then, chloroquine plus sulfadoxine/pyrimethamine was used. This therapy was subsequently replaced with artemether plus lumefantrine (AL). In 2017, three P. falciparum parasites harbouring C580Y were identified in Wewak, East Sepik. Population-genetic analysis using whole-genome and haplotypes of kelch13 flanking microsatellite markers suggested that the parasites harbouring C580Y in PNG did not migrate from Southeast Asia. Rather, they had emerged independently from another region in New Guinea [11]. Considering the aggressive increase in the C580Y harbouring parasites in GMS region, there is a growing concern that a similar phenomenon may occur in PNG. Therefore, it is essential to assess whether the population of parasites harbouring C580Y has increased in the total parasite population in PNG.

In addition to the issue of ART resistance, previous ex vivo drug study also found that most P. falciparum parasites were resistant to chloroquine despite the discontinuation of chloroquine use in the early 2010s [12]. This is contrary to the observations in many African countries where chloroquine susceptibility recovered years after discontinuation [13,14,15].

To evaluate whether the parasites harbouring the C580Y mutation have increased since the first emergence and whether chloroquine resistance persists, a molecular epidemiological study was performed in 2020 in Wewak, East Sepik. The results were analysed with previous results obtained in 2002, 2003, and 2016–2018 [11, 12, 16, 17], which were conducted with the same design, target populations, and site as the present study. The results showed a significant increase in C580Y proportion and potential recovery of chloroquine susceptibility.

Methods

Study design and site

This study was conducted at two clinics (Wirui Urban and Town) in January and February 2020 in Wewak District, East Sepik Province, PNG [12]. The study area comprises a lowland swamp along the coast. High transmission rates of malaria occur throughout the year, with seasonal fluctuations [18]. Four Plasmodium species (P. falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium malariae) were observed in this region, with P. falciparum predominant.

Ethical approval for the study was obtained from the Medical Research Ethical Committee of Juntendo University (No. 2017070) and the Medical Research Advisory Committee of the PNG National Department of Health (MRAC No.16.41).

Patients and blood collection

In both clinics, patients with suspected malarial symptoms were screened using the Rapid Diagnosis Test (RDT) (Carestart™ Malaria HRP2/pLDH COMBO, Access Bio Inc., NJ, USA). Patients > 1 year of age with Plasmodium-positive results were recruited for the study and were enrolled after obtaining informed consent from the patients or their guardians. Patients who met the criteria for severe malaria and pregnant women in 1st trimester were not included. Blood samples (100 µl) were obtained through finger prick and transferred onto ET31CHR chromatography filter paper (Whatman Limited, Kent, UK). After drying at room temperature, the samples were separated in a plastic bag and stored at − 20 °C. Thick and thin blood smears were prepared and stained with 2% Giemsa for 45 min for parasite counting. Parasitaemia was determined by counting 1000 erythrocytes on a thin smear or 200 leukocytes on a thick smear (parasitaemia < 0.1%). All P. falciparum positive patients were treated with the AL regimen according to national guidelines and were asked to visit the clinics to evaluate parasite positivity on day 3 of treatment.

Malaria PCR, genotyping of kelch13, pfcrt, and pfmdr1

Parasite DNA was extracted from one quarter of each blood spot using the QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany). Plasmodium falciparum positivity was confirmed by species-specific PCR as previously described [19]. Polymorphisms were determined through direct sequencing, including kelch13 (propeller domain amino acid positions 427–726), P. falciparum chloroquine resistance transporter gene (pfcrt; amino acid positions 72–76), and P. falciparum multidrug resistance-1 gene (pfmdr1; amino acid positions 86, 184, 1034, 1042, and 1246). The detailed protocol of the PCR analysis for each target gene is described in Additional file 1. Pfcrt K76T and pfmdr1 N86Y are associated with chloroquine resistance, and confer chloroquine resistance [20, 21]; the opposite trend has been reported for lumefantrine [22,23,24]. The allele proportion of kelch13, pfcrt, and pfmdr1 in 2020 was compared to that in 2002, 2003, and 2016–2018 [11, 12, 16]. The genotypes of kelch13 in 2018 were analysed using the same samples previously reported [12].

Statistical analysis

Statistical analysis was performed using R software (version 4.1.0), with the Chi-square test for trend (Cochran-Armitage trend test). Statistical significance was set at p < 0.05.

Results

Patients and Plasmodium sp. specific PCR

Among the 335 patients screened with RDT, 118 had positive results for Plasmodium (Fig. 1). Of these, species-specific PCR revealed that 13 were parasite negative and nine were other species, resulting in 96 patients with P. falciparum (Additional file 2). Two patients who received an intramuscular injection of artemether within two weeks prior to enrollment were excluded from further analysis. Finally, 94 samples were used for molecular analysis. There were no significant differences in the background characteristics of the enrolled patients between the two clinics (Table 1). The median age was 17 years old [Inter quartile range (IQR): 13, 26.5, range: 4–7]. The median parasitaemia was2.2 × 106/µl (IQR: 0.9 × 106, 5.6 × 106, range: 0.04 × 106–40.2 × 106). No significant difference was observed between patient number, age, sex, and average parasitaemia in each year (Additional file 3).

Fig. 1
figure1

Flow chart of the study

Table 1 Characteristics of studied patients

Frequency of polymorphisms in kelch13, pfcrt, and pfmdr1

The frequency of polymorphisms in kelch13, pfcrt, and pfmdr1 in 2020 are shown in Fig. 2. C580Y was the only mutation in kelch13 and was identified in six patients (6.4%). For pfcrt, there were two haplotypes, wild-type (CVMNK) and mutant (SVMNT; mutation underlined), with similar proportions (53.3% in wild-type and 46.7% in mutant). In pfmdr1, polymorphisms were observed at the amino acid positions 86, 184, and 1042; nearly all harboured the wild-type except the position 184.

Fig. 2
figure2

Changes in kelch13, pfcrt and pfmdr1 allele proportion in 2002, 2003, 2016–2018, and 2020. Each colour corresponds an allele type. Blue corresponds to wild-type, yellow to mix, and red to mutant

These results were compared with the previous results obtained in 2002, 2003, and 2016–2018 (Fig. 2) [11, 12, 16, 17]. All these studies were performed as passive case detections at the same clinics. In kelch13, since the first detection of C580Y in three patients (2.2%) in 2017 [11], there has been a statistically significant increase in the proportion of C580Y (p = 4.2 × 10–3). In both pfcrt and pfmdr1, the majority of parasites harboured chloroquine-resistant types in 2002 and 2003 [16]. However, a marked shift of allele proportion to chloroquine-sensitive types was observed after the mid-2010s (Fig. 2). In pfcrt, wild-type significantly increased from 1.9% in 2016 to 46.7% in 2020 (p = 8.9 × 10–16). In pfmdr1, the N86 allele also significantly increased from 59.5% in 2016 to 91.4% in 2020 (p = 2.3 × 10–6). The transition to the chloroquine-sensitive form of pfmdr1 occurred at least three years earlier than that for pfcrt. In fact, the proportion of the pfmdr1N86 allele in 2016 was higher than that of K76 in pfcrt in 2020. Although polymorphisms were observed at positions 184 and 1042, the proportion fluctuated annually without any upward or downward trends. Only the wild-type allele was found throughout the study period (2002–2003, 2016–2018, and 2020) at positions 1034 and 1246.

Follow-up of patients on day 3

Since the patients were asked for a voluntary visit on day 3, among the 94 patients treated with AL, 27 were followed-up with clinical assessment on day 3. Three patients were infected with the C580Y mutant. Only one patient was afebrile on day 3. Prevalent symptoms remaining on day 3 were headache (33%) and muscle/joint pain (15%) (Additional file 4). The smears of all follow-up patients showed an absence of parasites on day 3. There were no cases of early treatment failure.

Discussion

Ex vivo and molecular epidemiological study related to malaria drug resistance was conducted in 2002 and 2003 in East Sepik, PNG [16, 17]. After a long interval between 2004 and 2015, the study was restarted in 2016 [12]. In 2017, three kelch13 C580Y mutants were identified [11]. This raised concern that ART-resistant P. falciparum parasites may have already emerged and spread in the study area. In this study, the proportion of kelch13 C580Y was relatively low, but it had increased significantly since the first detection. In general, drug-resistant malaria increases slowly at the beginning of an emergence, but increases rapidly as the frequency of resistant parasites increases. Indeed, C580Y frequencies gradually increased approximately five years from the initial detection, but then rapidly expanded and even overtook the other kelch13 alleles in Cambodia and Western Thailand [7, 8, 25]. Therefore, even though the current proportion of kelch13 C580Y is low in PNG, rapid expansion in the near future could be anticipated.

Conversely, regional malaria epidemiological factors in PNG may suppress the rapid increase in ART-resistant parasites. First, residents in the study area developed higher levels of herd immunity to malaria than those in the GMS region because of the higher malaria transmission intensity in PNG [26, 27]. This can considerably influence the clearance of ART-resistant parasites from human hosts [28] and may slow the rate of increase in the C580Y allele in the region. Second, because ART is primarily used in ACT, the presence of resistant parasites to ACT partner drugs significantly affects the diffusion rate of C580Y. In the GMS region, parasites resistant to partner drug(s), mefloquine, and piperaquine have already emerged and spread [29, 30]. In particular, parasites harbouring both kelch13 C580Y and plasmepsin 2/3 copy number variants, which are molecular markers for piperaquine resistance, have rapidly increased in West Cambodia [25, 31]. In contrast, in PNG there is no evidence that parasites are resistant to lumefantrine, the currently used partner drug of ACT. Previous ex vivo drug susceptibility study from 2016 to 2018 also demonstrated that the average IC50 to lumefantrine was 4.6 nM and no parasite fulfilled the criteria of ex vivo lumefantrine resistance [12]. Furthermore, no patient exhibited parasite positivity on day 3, although the follow-up number was small.

The effects of the C580Y mutation on ART resistance are important determinants of the survival of drug-resistant parasites. However, the introduction of C580Y into P. falciparum clones did not substantially increase the level of in vitro ART resistance compared to other mutations, such as R539T [32]. In addition, drug-resistant mutations generally confer a decrease in parasite fitness, which often leads to a survival disadvantage [33,34,35,36]. Several laboratory studies have demonstrated that the growth rates of C580Y harbouring transgenic parasites were equal to or less than those of transgenic parasites with other kelch13 mutations, such as R561H, E252Q, and G538V. This suggest that C580Y incurs level of fitness impairment that is at least similar to that of the other kelch13 mutations [32, 37, 38]. These laboratory findings are inconsistent with the field observations in the GMS region, where the C580Y mutant outcompeted other mutants [7, 8, 25]. This implies that some unique background genetic changes in the South-East Asia (SEA) parasites play a beneficial role in the survival of the SEA parasites harbouring the C580Y allele. This might include compensation for the harmful effects of the C580Y mutation [37, 39]. Several single nucleotide polymorphisms have been identified in SEA kelch13 isolates [40]. However, among thesesingle nucleotide polymorphisms, only one (ferredoxin D193Y) was found in our PNG C580Y mutants [11], suggesting that PNG C580Y mutants do not possess the same background genetic changes as SEA C580Y mutants.

For chloroquine susceptibility, the average IC50s values were still high (80.5–106.6 nM) with a low proportion of pfcrt K76 wild-type (2.3–11.7%) during 2016–2018 [12]. In 2020, however, the proportion of pfcrt K76 wild-type rapidly increased to 46.7%. Since a significant association between the pfcrt K76 wild-type allele and ex vivo chloroquine susceptibility was confirmed [12], the observed rapid increase in the pfcrt K76 allele suggests that chloroquine sensitivity has been recovering. The significant increase in pfmdr1 N86 wild-type from 59.5% to 91.4% in the three years to 2020 also suggests the potential resurgence of chloroquine sensitivity. Although this phenomenon has been widely observed in African countries [13,14,15], it is very rare in SEA [41].

In the study area, decreased ex vivo susceptibility to lumefantrine was significantly associated with pfmdr1 N86 [12], consistent with a previous transfection study showing that an allelic change from N86Y to N86 increased IC50 for lumefantrine three to four times [24]. There are considerable reports that pfmdr1 N86 wild-type is selected by AL treatment [22, 23]. Furthermore, a recent meta-analysis of 60 AL clinical trials revealed that only 38% of patients treated with AL were symptomatic when the infection recurred [42]. If patients are asymptomatic at the time of recurrence, they are unlikely to seek treatment, resulting in the persistence of parasitaemia. This would increase the chance of parasites being transmitted to other human hosts, which could subsequently spread the drug-resistant mutations in the parasite population. In addition, a specific pfmdr1 haplotype (multicopy pfmdr1 in addition to N86 and Y184F) has increased and become predominant in Cambodia and Vietnam [43]. Gene editing experiments have shown that this haplotype significantly reduces parasite susceptibility to lumefantrine [43]. In the study site, although this haplotype was not detected [11], all these genetic changes (multicopy pfmdr1, N86, and Y184F) were individually observed. Therefore, in addition to the assessment of the relapse rate following AL, it is necessary to monitor the appearance of this haplotype.

Conclusions

Since its first identification in 2017, kelch13 C580Y harbouring P. falciparum parasites have been increasing in Wewak, East Sepik, PNG. A significant increase in pfcrt K76 and pfmdr1 N86 was also observed. This suggests a possible recovery of chloroquine sensitivity and, on the other hand, a decrease in sensitivity to lumefantrine, the ACT partner drug. The increasing frequency of kelch13 C580Y mutants under these circumstances is a warning sign that parasites resistant to AL will emerge in the near future. Thus, it is important to enhance continuous monitoring to detect early signs of the emergence of ACT-resistant parasites.

Availability of data and materials

The primary datasets used and analysed during the current study are available from the corresponding author upon reasonable request.

Abbreviations

ACT:

Artemisinin combination therapy

ART:

Artemisinin

AL:

Artemether plus lumefantrine

GMS:

Greater Mekong Subregion

IC50 :

50% Growth inhibitory concentration

IQR:

Inter quartile range

PCR:

Polymerase chain reaction

pfcrt :

P. falciparum chloroquine resistance transporter gene

pfmdr1 :

P. falciparum multidrug resistance-1 gene

PNG:

Papua New Guinea

RDT:

Rapid diagnostic test

SEA:

Southeast Asia

References

  1. 1.

    Murray CJ, Rosenfeld LC, Lim SS, Andrews KG, Foreman KJ, Haring D, et al. Global malaria mortality between 1980 and 2010: a systematic analysis. Lancet. 2012;379:413–31.

    PubMed  Article  PubMed Central  Google Scholar 

  2. 2.

    Noedl H, Se Y, Schaecher K, Smith BL, Socheat D, Fukuda MM. Evidence of artemisinin-resistant malaria in western Cambodia. N Engl J Med. 2008;359:2619–20.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  3. 3.

    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.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  4. 4.

    Imwong M, Suwannasin K, Kunasol C, Sutawong K, Mayxay M, Rekol H, et al. The spread of artemisinin-resistant Plasmodium falciparum in the Greater Mekong subregion: a molecular epidemiology observational study. Lancet Infect Dis. 2017;17:491–7.

    PubMed  PubMed Central  Article  Google Scholar 

  5. 5.

    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.

    PubMed  Article  CAS  Google Scholar 

  6. 6.

    WHO. World Malaria Report 2020. Geneva: World Health Organization; 2020.

    Google Scholar 

  7. 7.

    Anderson TJ, Nair S, McDew-White M, Cheeseman IH, Nkhoma S, Bilgic F, et al. Population parameters underlying an ongoing soft sweep in Southeast Asian malaria parasites. Mol Biol Evol. 2017;34:131–44.

    CAS  PubMed  Article  Google Scholar 

  8. 8.

    Imwong M, Hien TT, Thuy-Nhien NT, Dondorp AM, White NJ. Spread of a single multidrug resistant malaria parasite lineage (PfPailin) to Vietnam. Lancet Infect Dis. 2017;17:1022–3.

    PubMed  Article  Google Scholar 

  9. 9.

    Chenet SM, Akinyi Okoth S, Huber CS, Chandrabose J, Lucchi NW, Talundzic E, et al. Independent emergence of the Plasmodium falciparum kelch propeller domain mutant allele C580Y in Guyana. J Infect Dis. 2016;213:1472–5.

    CAS  PubMed  Article  Google Scholar 

  10. 10.

    Mathieu LC, Cox H, Early AM, Mok S, Lazrek Y, Paquet JC, et al. Local emergence in Amazonia of Plasmodium falciparum k13 C580Y mutants associated with in vitro artemisinin resistance. Elife. 2020;9:e51015.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. 11.

    Miotto O, Sekihara M, Tachibana SI, Yamauchi M, Pearson RD, Amato R, et al. Emergence of artemisinin-resistant Plasmodium falciparum with kelch13 C580Y mutations on the island of New Guinea. PLoS Pathog. 2020;16:e1009133.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. 12.

    Sekihara M, Tachibana SI, Yamauchi M, Yatsushiro S, Tiwara S, Fukuda N, et al. Lack of significant recovery of chloroquine sensitivity in Plasmodium falciparum parasites following discontinuance of chloroquine use in Papua New Guinea. Malar J. 2018;17:434.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. 13.

    Mita T, Kaneko A, Lum JK, Bwijo B, Takechi M, Zungu IL, et al. Recovery of chloroquine sensitivity and low prevalence of the Plasmodium falciparum chloroquine resistance transporter gene mutation K76T following the discontinuance of chloroquine use in Malawi. Am J Trop Med Hyg. 2003;68:413–5.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  14. 14.

    Laufer MK, Thesing PC, Eddington ND, Masonga R, Dzinjalamala FK, Takala SL, et al. Return of chloroquine antimalarial efficacy in Malawi. N Engl J Med. 2006;355:1959–66.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  15. 15.

    Balikagala B, Sakurai-Yatsushiro M, Tachibana SI, Ikeda M, Yamauchi M, Katuro OT, et al. Recovery and stable persistence of chloroquine sensitivity in Plasmodium falciparum parasites after its discontinued use in Northern Uganda. Malar J. 2020;19:76.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. 16.

    Mita T, Kaneko A, Hombhanje F, Hwaihwanje I, Takahashi N, Osawa H, et al. Role of pfmdr1 mutations on chloroquine resistance in Plasmodium falciparum isolates with pfcrt K76T from Papua New Guinea. Acta Trop. 2006;98:137–44.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  17. 17.

    Mita T, Kaneko A, Hwaihwanje I, Tsukahara T, Takahashi N, Osawa H, et al. Rapid selection of dhfr mutant allele in Plasmodium falciparum isolates after the introduction of sulfadoxine/pyrimethamine in combination with 4-aminoquinolines in Papua New Guinea. Infect Genet Evol. 2006;6:447–52.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  18. 18.

    Muller I, Bockarie M, Alpers M, Smith T. The epidemiology of malaria in Papua New Guinea. Trends Parasitol. 2003;19:253–9.

    PubMed  Article  PubMed Central  Google Scholar 

  19. 19.

    Rubio JM, Benito A, Berzosa PJ, Roche J, Puente S, Subirats M, et al. Usefulness of seminested multiplex PCR in surveillance of imported malaria in Spain. J Clin Microbiol. 1999;37:3260–4.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. 20.

    Fidock DA, Nomura T, Talley AK, Cooper RA, Dzekunov SM, Ferdig MT, et al. Mutations in the P. falciparum digestive vacuole transmembrane protein PfCRT and evidence for their role in chloroquine resistance. Mol Cell. 2000;6:861–71.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  21. 21.

    Foote SJ, Kyle DE, Martin RK, Oduola AM, Forsyth K, Kemp DJ, et al. Several alleles of the multidrug-resistance gene are closely linked to chloroquine resistance in Plasmodium falciparum. Nature. 1990;345:255–8.

    CAS  PubMed  Article  Google Scholar 

  22. 22.

    Sisowath C, Strömberg J, Mårtensson A, Msellem M, Obondo C, Björkman A, et al. In vivo selection of Plasmodium falciparum pfmdr1 86N coding alleles by artemether-lumefantrine (Coartem). J Infect Dis. 2005;191:1014–7.

    CAS  PubMed  Article  Google Scholar 

  23. 23.

    Sisowath C, Petersen I, Veiga MI, Mårtensson A, Premji Z, Björkman A, et al. In vivo selection of Plasmodium falciparum parasites carrying the chloroquine-susceptible pfcrt K76 allele after treatment with artemether-lumefantrine in Africa. J Infect Dis. 2009;199:750–7.

    CAS  PubMed  Article  Google Scholar 

  24. 24.

    Veiga MI, Dhingra SK, Henrich PP, Straimer J, Gnädig N, Uhlemann AC, et al. Globally prevalent PfMDR1 mutations modulate Plasmodium falciparum susceptibility to artemisinin-based combination therapies. Nat Commun. 2016;7:11553.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  25. 25.

    Imwong M, Dhorda M, Myo Tun K, Thu AM, Phyo AP, Proux S, et al. Molecular epidemiology of resistance to antimalarial drugs in the Greater Mekong subregion: an observational study. Lancet Infect Dis. 2020;20:1470–80.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  26. 26.

    Rogerson SJ, Wijesinghe RS, Meshnick SR. Host immunity as a determinant of treatment outcome in Plasmodium falciparum malaria. Lancet Infect Dis. 2010;10:51–9.

    CAS  PubMed  Article  Google Scholar 

  27. 27.

    Kattenberg JH, Gumal DL, Ome-Kaius M, Kiniboro B, Philip M, Jally S, et al. The epidemiology of Plasmodium falciparum and Plasmodium vivax in East Sepik Province, Papua New Guinea, pre- and post-implementation of national malaria control efforts. Malar J. 2020;19:198.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. 28.

    Ataide R, Ashley EA, Powell R, Chan JA, Malloy MJ, O’Flaherty K, et al. Host immunity to Plasmodium falciparum and the assessment of emerging artemisinin resistance in a multinational cohort. Proc Natl Acad Sci USA. 2017;114:3515–20.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  29. 29.

    Spring MD, Lin JT, Manning JE, Vanachayangkul P, Somethy S, Bun R, et al. Dihydroartemisinin–piperaquine failure associated with a triple mutant including kelch13 C580Y in Cambodia: an observational cohort study. Lancet Infect Dis. 2015;15:683–91.

    CAS  PubMed  Article  Google Scholar 

  30. 30.

    van der Pluijm RW, Imwong M, Chau NH, Hoa NT, Thuy-Nhien NT, Thanh NV, et al. Determinants of dihydroartemisinin–piperaquine treatment failure in Plasmodium falciparum malaria in Cambodia, Thailand, and Vietnam: a prospective clinical, pharmacological, and genetic study. Lancet Infect Dis. 2019;19:952–61.

    PubMed  PubMed Central  Article  Google Scholar 

  31. 31.

    Amato R, Lim P, Miotto O, Amaratunga C, Dek D, Pearson RD, et al. Genetic markers associated with dihydroartemisinin–piperaquine failure in Plasmodium falciparum malaria in Cambodia: a genotype–phenotype association study. Lancet Infect Dis. 2017;17:164–73.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  32. 32.

    Nair S, Li X, Arya GA, McDew-White M, Ferrari M, Nosten F, et al. Fitness costs and the rapid spread of kelch13-C580Y substitutions conferring artemisinin resistance. Antimicrob Agents Chemother. 2018;62:e00605-e618.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  33. 33.

    Mackinnon MJ, Hastings IM. The evolution of multiple drug resistance in malaria parasites. Trans R Soc Trop Med Hyg. 1998;92:188–95.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  34. 34.

    Hastings IM, Donnelly MJ. The impact of antimalarial drug resistance mutations on parasite fitness, and its implications for the evolution of resistance. Drug Resist Updat. 2005;8:43–50.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  35. 35.

    Walliker D, Hunt P, Babiker H. Fitness of drug-resistant malaria parasites. Acta Trop. 2005;94:251–9.

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  36. 36.

    Rosenthal PJ. The interplay between drug resistance and fitness in malaria parasites. Mol Microbiol. 2013;89:1025–38.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  37. 37.

    Straimer J, Gnadig NF, Stokes BH, Ehrenberger M, Crane AA, Fidock DA. Plasmodium falciparum K13 mutations differentially impact ozonide susceptibility and parasite fitness in vitro. mBio. 2017;8:e00172-17.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. 38.

    Tirrell AR, Vendrely KM, Checkley LA, Davis SZ, McDew-White M, Cheeseman IH, et al. Pairwise growth competitions identify relative fitness relationships among artemisinin resistant Plasmodium falciparum field isolates. Malar J. 2019;18:295.

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  39. 39.

    Stokes BH, Dhingra SK, Rubiano K, Mok S, Straimer J, Gnadig NF, et al. Plasmodium falciparum K13 mutations in Africa and Asia impact artemisinin resistance and parasite fitness. Elife. 2021;10:e66277.

    PubMed  PubMed Central  Article  Google Scholar 

  40. 40.

    Miotto O, Amato R, Ashley EA, MacInnis B, Almagro-Garcia J, Amaratunga C, et al. Genetic architecture of artemisinin-resistant Plasmodium falciparum. Nat Genet. 2015;47:226–34.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. 41.

    Liu DQ, Liu RJ, Ren DX, Gao DQ, Zhang CY, Qui CP, et al. Changes in the resistance of Plasmodium falciparum to chloroquine in Hainan. China Bull World Health Organ. 1995;73:483–6.

    CAS  PubMed  Google Scholar 

  42. 42.

    Mumtaz R, Okell LC, Challenger JD. Asymptomatic recrudescence after artemether-lumefantrine treatment for uncomplicated falciparum malaria: a systematic review and meta-analysis. Malar J. 2020;19:453.

    PubMed  PubMed Central  Article  Google Scholar 

  43. 43.

    Calcada C, Silva M, Baptista V, Thathy V, Silva-Pedrosa R, Granja D, et al. Expansion of a specific Plasmodium falciparum PfMDR1 haplotype in Southeast Asia with increased substrate transport. mBio. 2020;11:e02093-20.

    CAS  PubMed  PubMed Central  Article  Google Scholar 

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Acknowledgements

We thank all the study participants and their guardians. We are grateful to Steven Tiwara, Charlie Amai, Alphonse Coll, John Sambi and Douglas Tambi, and staff from Wewak Clinic, Town Clinic, for their kind cooperation in the field. We also thank Shuxin Song for technical assistance.

Funding

This study was financially supported by Grants-in-aid for scientific research [26460515, 17H04074] awarded to Professor Toshihiro Mita.

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Affiliations

Authors

Contributions

NY and TM designed and coordinated the study; MY, ST, and FH performed the field study; NY and RM performed the laboratory work; NY and TM analysed and interpreted the data; NY and TM wrote the manuscript. All the authors contributed significantly to this work. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Toshihiro Mita.

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Ethics approval and consent to participate

Ethical approval was obtained from the Medical Research Ethical Committee of Juntendo University (No. 2017070) and the Medical Research Advisory Committee of the Papua New Guinea National Department of Health (MRAC No. 16.41).

Consent for publication

Prior to participation, all study subjects consented to the publication of study results in the medical literature in an anonymized manner.

Competing interests

All authors declare no competing interests.

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Supplementary Information

Additional file 1:

Primers and protocol for the PCR analysis.

Additional file 2:

 Molecular diagnosis of enrolled samples.

Additional file 3:

 Background characteristics of enrolled patients each year (2002, 2003, and 2016–2018).

Additional file 4:

 Characteristics of studied patients (Day 3).

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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. Malar J 20, 410 (2021). https://doi.org/10.1186/s12936-021-03933-6

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Keywords

  • Plasmodium falciparum
  • Artemisinin
  • Chloroquine
  • Lumefantrine
  • Resistance
  • kelch13
  • pfcrt
  • pfmdr1
  • C580Y
  • Papua New Guinea