Background
Malaria is still one of the three major infectious diseases worldwide with 216 million cases and 445,000 deaths over 100 countries in 2016 [
1]. Although new sustainable development goals have proposed to end malaria epidemic by 2030 [
2], emergence and spread of drug-resistant parasites could be a major obstacle for this achievement.
Plasmodium falciparum parasites resistant to artemisinin-based combination therapy (ACT), the current first-line treatment for uncomplicated malaria, have already spread across the Greater Mekong sub-region [
3]. However, licensed anti-malarial drugs that possess similar levels of efficacy as artemisinins have not yet been obtained. Under such circumstances, an approach that rotates licensed anti-malarial drugs is suggested to be a potential strategy to combat drug-resistant parasites. Chloroquine is a candidate drug that is potentially applicable to such a strategy. This is because chloroquine-susceptible parasites have outcompeted the resistant parasites and have expanded in the absence of chloroquine selecting pressure [
4‐
6]; subsequently, chloroquine susceptibility has been recovered several years after its discontinuance in many endemic regions, particularly in Africa [
7‐
13]. However, a lack of this phenomenon has been also reported in some African and other endemic regions [
14‐
16]. Therefore, the extent of this reversal across malaria-endemic countries is not fully understood [
17].
In Papua New Guinea, since the first report of chloroquine-resistant
P. falciparum parasite in the 1970s [
18], resistant parasites have spread across the area. The clinical efficacy of chloroquine reached unacceptable levels by the mid-1990s [
19,
20]. In 2000, a combination regimen of chloroquine or amodiaquine plus sulphadoxine–pyrimethamine was introduced as a first-line treatment for uncomplicated malaria. However, treatment failure of these regimens against
P. falciparum reached 11–29% during 2003–2005 as assessed at day 28 [
21], and 15% during 2005–2007 [
22]. In 2010, chloroquine was completely removed from the official treatment regime and artemether plus lumefantrine was officially introduced as a first-line regimen for uncomplicated malaria. Following this discontinuance, change in the average 50% growth inhibitory concentration (IC
50) to chloroquine was reported as 167 nM during 2005–2007 to 87 nM during 2011–2013 in the Madang Province [
23]. However, this IC
50 value was still much higher than those reported in regions with reversal of chloroquine susceptibility such as Kenya (22.4 nM) [
12] and Senegal (34.8 nM) [
10]. Additionally, almost all parasites in the Madang study still harboured a chloroquine-resistant allele (K76T mutation) in the
P. falciparum chloroquine-resistance transporter (
pfcrt). These results indicate that a complete recovery of chloroquine susceptibility after its withdrawal has not been evidenced in Papua New Guinea and warrants further investigation. An ex vivo study was therefore performed in 2016–2018, 6–8 years after chloroquine withdrawal in Wewak district, East Sepik Province, in which the ex vivo drug susceptibility study was previously conducted during 2002–2003 [
24].
Discussion
There are dozens of epidemiological studies showing that chloroquine-susceptible parasites replace resistant parasites in the absence of chloroquine selection [
7‐
13]. However, the present analysis revealed a lack of substantial recovery of chloroquine susceptibility at 6–8 years after the withdrawal of chloroquine in Papua New Guinea.
In nearly all endemic regions where chloroquine-sensitive parasites re-emerged, reduction of parasites harbouring a K76T mutation in
pfcrt played a pivotal role towards this phenomenon [
7‐
10,
12], though some exclusive regions have been reported, such as French Guiana [
39]. This is because the K76T mutation imposes some fitness cost to the parasites [
40‐
43]. A reverse genetic study evidenced that introduction of K76T into chloroquine-susceptible clones induced a reduction in the growth rate [
41,
42]. One suggested mechanism for this is that K76T harbouring parasites show functional impairment of haemoglobin digestion, which subsequently reduces the supply of amino acids required for parasite growth [
41]. Fitness reduction of chloroquine-resistant parasites was also reported in mosquito stages; K76T-bearing parasites were less selected than K76-bearing parasites in
Anopheles arabiensis [
40]. Because of these disadvantages, K76T-harbouring parasites have been outcompeted by K76-harbouring parasites in the absence of chloroquine pressure [
4,
6]. In this study,
pfcrt K76T harbouring parasites showed a significantly higher IC
50s than those in
pfcrt K76 harbouring parasites. This observation is same as those observed in African endemic regions [
11,
12], suggesting that an associated mechanism of chloroquine resistance would be common in parasites in Africa and Papua New Guinea. However, the majority of parasites still harboured the K76T allele and recovery of chloroquine susceptibility has not been observed even after withdrawal of chloroquine use. These observations suggest that genetic change(s) other than K76T in
pfcrt and/or other unknown gene(s) compensate the fitness cost imposed by K76T and may explain the reason why chloroquine susceptibility is not returning at the same rate in Africa. It could be also possible to conjecture that, in Africa, there are some particular K76-harbouring parasites which have some stronger fitness advantage than K76-harbouring parasites in Papua New Guinea.
It has been suggested that amino acid differences flanking K76T affect the fitness disadvantage imposed by K76T [
42]. In the natural parasite population, there are two major mutant haplotypes constructed by five amino acids at positions 72–76: CV
IET and
SVMN
T [
42,
44]. In Papua New Guinea, nearly all mutant parasites harboured a
SVMN
T haplotype, and the CV
IET haplotype was also observed with extremely low prevalence [
45,
46]. A transfection study has reported that a
SVMN
T introduced isolate depicted lower growth rates than a wild-type (CVMNK) isolate, but better growth rate than the CV
IET introduced isolate [
42]. A quick repopulation of K76-harbouring parasites after chloroquine discontinuance has mostly been observed in the CV
IET haplotype regions. Therefore, the fact that all
pfcrt mutants harboured a
SVMN
T haplotype may partly explain the persistent high prevalence of K76T in this study region.
However, it is striking that K76-harbouring parasites significantly increased during 2016–2018. This is the first study to show the potential repopulation of K76 harbouring parasites after chloroquine withdrawal in a
SVMN
T prevalent region. Despite a significant increase, the majority of parasites still harboured the K76T allele. Many environmental, population genetic, and parasitological factors potentially affect the rate of repopulation of susceptible parasites after chloroquine discontinuance [
5,
47,
48]. The frequency of susceptible parasites in the parasite population when chloroquine pressure was removed is one such important factor. Historically, the K76T allele had already become predominant or was nearly fixed by the late 1990s in many endemic regions in Papua New Guinea [
45,
49‐
51]. Accordingly, the K76T prevalence in our study region reached around 95% during 2002–2003 [
24]. Considering the strong selection pressure posed by the use of chloroquine for the treatment of uncomplicated malaria before 2010, an extremely low frequency of susceptible parasites is expected at the time when chloroquine was withdrawn. Therefore, it is considered that the observed high proportion of K76T harbouring parasites may be partly explained by the presumed extremely low initial proportion of K76-harbouring parasites.
A requirement for secondary determinants has been suggested for the augmentation of chloroquine resistance [
52‐
54]. One such candidate gene is
pfmdr1 [
35,
53]. In the present study, parasites with Y184F mutation displayed a significantly higher IC
50 for chloroquine compared to those with Y184. This association was also found in parasites bearing
pfcrt-K76T, suggesting that Y184F confers an additional factor for decreased chloroquine susceptibility in our study area. However, a previous reverse genetic study reported that an allele change from Y184 to Y184F conferred only a slight decrease in chloroquine susceptibility in a laboratory clone harbouring
pfcrt-
SVMN
T [
55]. One possible explanation for this discrepancy is that genetic background could influence the role of the Y184F mutation on the augmentation of chloroquine resistance. The parasite clone used in the study by Veiga et al., was a KC5 clone, a progeny of the genetic cross between 7G8 (Brazil) and GB4 (Ghana) parasites [
56].
Persistence of chloroquine-selecting pressure potentially interferes the recovery of chloroquine-sensitive parasites. In Lagos, Nigeria where chloroquine was still widely used even after the introduction of ACT,
P. falciparum parasites harbouring a K76T mutation continued to be highly prevalent [
57]. In Papua New Guinea, however, ACT has been used as a first-line treatment for all malaria species including
P. vivax. Chloroquine has not been included in the official malaria-treatment regimen. However, although no stock of chloroquine in clinics and hospitals was confirmed in the studied area, chloroquine was still sold at two private pharmacies with a cheaper price than other anti-malarial drugs throughout the study period. Indeed, 2–4% of enrolled patients used chloroquine before visiting the clinics in this study. These observations indicate that chloroquine is still in use by some patients, which could play some role in a result of lack of complete withdrawal of chloroquine.
For lumefantrine, our average IC
50 values (4.6 nM) were higher than those (1.5 nM) reported in Madang district during 2011–2013 [
23]. The N86 allele frequencies in our study (59–74%) were also much higher than those in the Madang study (< 10%). In our study, a significant association was detected between higher IC
50 values for lumefantrine and the N86 allele in
pfmdr1. This is consistent with the previous transfection study in which an allelic change from N86Y to N86 resulted in a three to fourfold increase in the IC
50 for lumefantrine [
55]. A recent meta-analysis has also shown that patients infected with parasites harbouring N86 had a fivefold risk of recrudescence in following artemether/lumefantrine treatment compared to those infected with parasites harbouring N86Y [
58]. The observed lower lumefantrine susceptibility and higher
pfmdr1-N86 prevalence than that in the previous observation [
23] may raise the possibility of a decreasing trend of lumefantrine susceptibility in Papua New Guinea.
Authors’ contributions
MS, FH, and TMita designed and coordinated the study; MS, SIT, MY, SY, ST, TMori, and TMita performed the field study; ST, NF, MI, and MH performed the laboratory work; MS and TMita analysed and interpreted the data; MS and TMita wrote the manuscript. All authors contributed significantly to this work. All authors read and approved the final manuscript.