Increased in the Prevalence of Plasmodium Falciparum With Kelch13 C580Y Mutations and the Decline in pfcrt and pfmdr1 Mutant Alleles in Papua New Guinea

The C580Y mutation in Plasmodium falciparum kelch13 (pfk13) is the most commonly observed variant in artemisinin-resistant isolates in the Greater Mekong Subregion (GMS). Until 2017, it had not been identied outside GMS, except for Guyana. In 2017, we identied three parasites carrying the C580Y mutation 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.


Abstract Background
The C580Y mutation in Plasmodium falciparum kelch13 (pfk13) is the most commonly observed variant in artemisinin-resistant isolates in the Greater Mekong Subregion (GMS). Until 2017, it had not been identi ed outside GMS, except for Guyana. In 2017, we identi ed three parasites carrying the C580Y mutation 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, we conducted a cross-sectional survey 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 pfk13, pfcrt, and pfmdr1 were determined. Parasite positivity was examined on day 3.

Conclusions
Under the conditions of signi cant increases in pfcrt K76 and pfmdr1 N86 alleles in PNG, the increase in pfk13 C580Y mutants may be a warning indicator of the emergence of parasites resistant to the currently used rst-line treatment regimen of artemether plus lumefantrine. Therefore, nationwide surveillance of molecular markers for drug resistance and assessment of its therapeutic effect, are important. Background Artemisinin (ART)-based combination therapy (ACT) is a widely used rst-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 P. falciparum was rst reported in the Greater Mekong Subregion (GMS) in 2006 [2][3][4]. Since then, ART-resistant parasites have rapidly spread in the region, partly because of the emergence of resistance to partner drug(s) of ACT [5,6]. Therefore, the emergence and spread of RT-resistant parasites outside the GMS has become a global concern.
Propeller polymorphisms of the pfk13 gene are useful molecular markers to monitor the emergence and spread of ART resistance [4,7]. To date, ten or more non-synonymous mutations in pfk13 have been validated as polymorphisms for ART resistance. These include N458Y, Y493H, R539T, I543T, R561H, and C580Y [8]. In particular, C580Y has gradually outcompeted the other mutations and become dominant in some parts of the GMR region (GMSR) [5,9,10]. C580Y is considered the most useful molecular marker to trace the spread of ART resistance in GMS. However, outside the GMSR, this mutation has been detected only in Guyana [11,12] and, as we reported more recently, in Papua New Guinea (PNG) [13].
In PNG, chloroquine plus sulfadoxine/pyrimethamine had been adopted as the o cial rst-line treatment regimen for uncomplicated malaria by 2010. This therapy was subsequently replaced with artemether plus lumefantrine (AL). In 2017, we identi ed three P. falciparum parasites harboring C580Y in Wewak, East Sepik. Population-genetic analysis using whole-genome and haplotypes of pfk13 anking microsatellite markers suggested that the C580Y in PNG did not migrate from Southeast Asia. Rather, it had independently emerged from another region in New Guinea [13]. Considering the aggressive increase in the C580Y harboring parasites in GMSR, there is a growing concern that a similar phenomenon may occur in PNG. Therefore, it is essential to assess whether parasites harboring C580Y have increased in the parasite population in PNG.
To evaluate whether levels of ART-resistant molecular markers have increased since the rst emergence and whether chloroquine resistance persists, a molecular epidemiological study was performed in 2020 in Wewak, East Sepik. Ex vivo drug susceptibility studies had previously been conducted there in 2002, 2003, and 2016-2018 [13,14,[29][30][31]. The results we describe show a signi cant increase in C580Y prevalence and the potential recovery of chloroquine susceptibility.

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 [14]. The study area comprises a lowland swamp along the coast. High transmission rates of malaria occur throughout the year, with seasonal uctuations [32]. All four Plasmodium species for human malaria were observed in this region, with P.

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. Blood samples (100 µl) were obtained by nger prick and transferred onto ET31CHR chromatography lter 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. 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.

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 signi cance was set at p < 0.05.

Results
Patients and Plasmodium sp. speci c PCR Among the 335 patients screened with RDT, 118 had positive results for Plasmodium (Fig. 1). Of these, species-speci c PCR revealed that 13 were parasite negative and nine were other species, resulting in 96 patients with P. falciparum (Additional le 1). 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 signi cant differences in the background characteristics of the enrolled patients between the two clinics ( Table 1). The median age was 17 years and the median parasitemia was 0.74%. No signi cant difference was observed between patient number, age, sex, and average parasitemia in each year (Additional le 2). C580Y was the only mutation observed in pfk13 and was identi ed in six patients (5.2%) in 2018 and six (6.4%) in 2020 (Fig. 2). Since the rst detection of C580Y in three patients (2.2%) in 2017 [13], there has been a statistically signi cant increase in the prevalence of C580Y (p = 4.2 × 10 − 3 , Cochran-Armitage trend test).
In both pfcrt and pfmdr1, the majority of parasites harbored chloroquine resistant types in 2002 and 2003 [29]. However, a marked shift of allele prevalence to chloroquine-sensitive types was observed after the mid-2010s (Fig. 2). In pfcrt, there were two haplotypes: wild-type (CVMNK) and mutant (SVMNT; mutation underlined). Wild-type prevalence signi cantly increased from 1.9% in 2016 to 46.7% in 2020 (p = 8.9×10 − 16 , Cochran-Armitage trend test). In pfmdr1, the N86 allele also signi cantly increased from 59.5% in 2016 to 91.4% in 2020 (p = 2.3×10 − 6 , Cochran-Armitage trend test). The transition to the chloroquine-sensitive form of pfmdr1 occurred at least three years earlier than that of pfcrt. In fact, the prevalence of the pfmdr1 N86 allele in 2016 was higher than that of K76 in pfcrt in 2020. Although polymorphisms were observed at positions 184 and 1042, the prevalence uctuated annually without any upward or downward trends. Only the wild-type allele was found throughout the study period at positions 1034 and 1246.

Follow-up of patients on day 3
Among the 94 patients treated with AL, 27 were successfully followed-up with clinical assessment on day 3. The patients included three 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%). All follow-up patients showed an absence of parasites on day 3 smears. There were no cases of early treatment failure.

Discussion
We commenced an epidemiological study related to malaria drug resistance in 2002 in East Sepik, PNG [29,30]. After a long interval from 2004 to 2015, the study was restarted in 2016 [14]. In 2017, we identi ed three pfk13 C580Y mutants [13]. This raised concern that ART-resistant P. falciparum parasites may have already emerged and spread in the study area. In this study, the prevalence of pfk13 C580Y was relatively low, but it had increased signi cantly since the rst 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 ve years from the initial detection, but then rapidly expanded and even overtook the other pfk13 alleles in Cambodia and Western Thailand [5,9,10]. Therefore, even though the current prevalence of pfk13 C580Y is low in PNG, rapid expansion in the near future could be anticipated.
On the other hand, regional malaria epidemiological factors in PNG may suppress the rapid increase in ART-resistant parasites. First, residents in our study area developed higher levels of herd immunity to malaria than those in the GMSR because of higher malaria transmission intensity in PNG [34,35]. This can considerably in uence the clearance of ART-resistant parasites from human hosts [36] 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 signi cant affects the diffusion rate of C580Y. In the GMSR, parasites resistant to partner drug(s), me oquine, and piperaquine have already emerged and spread [37,38]. In particular, parasites harboring both pfk13 C580Y and plasmepsin 2/3 copy number variants, which are molecular markers for piperaquine resistance, have rapidly increased in West Cambodia [5,39]. In contrast, in PNG there is no evidence that parasites are resistant to lumefantrine, the currently used partner drug of ACT. Our previous ex vivo drug susceptibility study from 2016 to 2018 also demonstrated that the average IC 50 to lumefantrine was 4.6 nM and no parasite ful lled the criteria of ex vivo lumefantrine resistance [14]. Furthermore, we found that 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 drugresistant 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 [40]. In addition, drug-resistant mutations generally confer a decrease in parasite tness, which often leads to a survival disadvantage [41][42][43][44]. Several laboratory studies have demonstrated that the growth rates of C580Y harboring transgenic parasites were equal to or less than those of transgenic parasites with other pfk13 mutations, such as R561H, E252Q, and G538V, suggesting that C580Y incurs at least a similar level of tness impairment to the other pfk13 mutations [40,45,46]. These laboratory ndings are inconsistent with the eld observations in the GMSR where the C580Y mutant has outcompeted other mutants [5,9,10]. This implies that some unique background genetic changes in the South-East Asia (SEA) parasites play a bene cial role in the survival of the SEA parasites harboring the C580Y allele. This might include compensation for the harmful effects of the C580Y mutation. Several single nucleotide polymorphisms (SNPs) have been identi ed in SEA pfk13 isolates [47]. However, among these SNPs, only one (ferredoxin D193Y) was found in our PNG C580Y mutants [13], suggesting that PNG C580Y mutants do not possess the same background genetic changes as SEA C580Y mutants.
We previously found that decreased ex vivo susceptibility to lumefantrine was signi cantly associated with pfmdr1 N86 [14], consistent with a previous transfection study showing that an allelic change from N86Y to N86 increased IC 50 for lumefantrine three to four times [49]. Thereare considerable reports that pfmdr1 N86 wild-type is selected by AL treatment [50,51]. 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 [52]. If patients are asymptomatic at the time of recurrence, they are unlikely to seek treatment, resulting in the persistence of parasitemia. This would increase the chance that the parasites could be transmitted to other human hosts, which could subsequently spread the drug-resistant mutations in the parasite population. In addition, a speci c pfmdr1 haplotype (multicopy pfmdr1 in addition to N86 and Y184F) has increased and become predominant in Cambodia and Vietnam [53]. Gene editing experiments showed that this haplotype signi cantly reduces parasite susceptibility to lumefantrine [53]. In our study site, although this haplotype was not detected [13], 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 rst identi cation in 2017, pfk13 C580Y harboring P. falciparum parasites have been increasing in Wewak, East Sepik, PNG. A signi cant 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 pfk13 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.

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

Availability of data and materials
The primary datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

Competing interests
All authors declare no competing interests.

Funding
This study was nancially supported by Grants-in-aid for scienti c research [26305015, 17H04074,18KK0231] from Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan awarded to Professor Toshihiro Mita.
Authors' contributions NY and TM designed and coordinated the study; MY, ST, and FH performed the eld study; NY and RM performed the laboratory work; NY and TM analyzed and interpreted the data; NY and TM wrote the manuscript. All the authors contributed signi cantly to this work. All authors read and approved the nal manuscript.  Figure 1 Flow chart of the study