African isolates show a high proportion of multiple copies of the Plasmodium falciparum plasmepsin-2 gene, a piperaquine resistance marker

Emergence of Plasmodium falciparum resistance to anti-malarial drugs is currently the primary rationale supporting the development of new and well-tolerated drugs. While the estimated number of malaria cases in the world decreased from 237 million (218–278 million) in 2010, to 211 million (192–257 million) in 2015, the morbidity and the mortality have stabilized in 2016 with estimates of 216 million cases (196–263 million) and 445,000 deaths (compared to 446,000 in 2015) as reported by the World Health Organization (WHO) [1–3].

Globally, the vast majority of deaths (> 90%) caused by malaria is due to P. falciparum infections, occurring in Africa, in children under 5 years of age. Artemisinin-based combination therapy (ACT) which are currently recommended as first-line treatment of uncomplicated falciparum malaria, is less effective in Southeast Asia, particularly in Cambodia, where high rates of treatment failure associated with artemisinin and piperaquine resistance are currently reported [4–16]. The containment and the elimination of these multidrug resistant parasites in Southeast Asia are a priority for the WHO to avoid their spread to Africa as was the case with previous generations of anti-malarial drugs (e.g. chloroquine, sulfadoxine–pyrimethamine) [17]. Fortunately, molecular markers associated with such resistance are available [10]. In particular, mutations in the propeller domain of a kelch gene located on the chromosome 13 (kelch13), and amplification of a cluster of genes encoding both plasmepsin 2 (Pfpm2) and plasmepsin 3 proteins, have been recently shown to be associated with artemisinin and PPQ resistance, respectively [18–20].

According to the latest WHO update on artemisinin resistance [21], to be validated a kelch13 resistance mutant has to be correlated with delayed parasite clearance in clinical studies and reduced drug in vitro susceptibility with survival rate ≥ 1% expressed by the Ring-stage Survival Assay, (RSA0–3 h) in fresh isolates (ex vivo assays), or culture-adapted field parasites or kelch13 genome-edited parasites (in vitro assays) [22–25]. To date, only nine kelch13 mutations have been shown to be validated (C580Y, Y493H, R539T, I543T, N458Y, P533L, M476I, R561H and F446I). The F446I mutant is highly prevalent in Myanmar as recently reported [26]. In Africa, a broad array of rare non-synonymous mutations in the kelch13 gene have been described in P. falciparum isolates, but none of these mutants have been associated with artemisinin resistance [27], attesting that not all non-synonymous kelch13 mutations confer resistance to artemisinin.

More recently, resistance to PPQ has been associated with an increase of survival rates of parasite exposed to 200 nM PPQ for 48 h in the piperaquine survival assay (PSA) and with the amplification of plasmepsin 2–3 genes (Pfpm2–3) [6, 20]. In Cambodia, where high rates of treatment failure to dihydroartemisinin–piperaquine (DHA–PPQ) are observed (i.e. > 60% in some provinces), it has been demonstrated that amplification of Pfpm2 gene and presence of validated kelch13 mutations were highly predictive of DHA–PPQ treatment failure [20]. Most of these parasites harbour a single copy of Pfmdr1 gene leading to the recovery of mefloquine sensitivity [4, 6] and suggesting a natural antagonism between PPQ resistance and mefloquine resistance. However, so far it was not understood whether Pfmdr1 de-amplification (from multiple copies to single copy Pfmdr1) was due to the implementation of DHA–PPQ as first-line treatment or due to the release of mefloquine pressure and an increase in parasite fitness accompanying Pfmdr1 gene de-amplification. To date, DHA–PPQ resistance was confined to Southeast Asia. So far, only few studies conducted on parasites from Mozambique and Mali have provided evidence of the presence (at low frequency, 1.1%, 10%) of parasites carrying multiple copies of Pfpm2 [28, 29].

Facing the threat of losing all current artemisinin-based combinations front-line therapies due to resistance, a new generation of endoperoxides with more favourable pharmacokinetic profiles like the ozonide Artefenomel^® (OZ439) have been developed [30]. The efficacy of this new chemical entity was evaluated in combination with PPQ in African and Southeast Asian (Vietnam) patients with uncomplicated falciparum malaria infection [30]. The primary objective of this phase 2b clinical study was to determine whether a single oral dose combination of artefenomel/PPQ was efficacious and safe [≥ 95% of patients cured on the basis of polymerase chain reaction (PCR)-adjusted adequate clinical parasitological response at day 28 (ACPR28)] in adults and children infected by P. falciparum. Blood samples collected in 2014–2015 from this clinical trial offered the opportunity to investigate the proportion of multidrug resistant parasites (i.e. P. falciparum kelch13 mutants and gene copy number of both Pfpm2 and Pfmdr1). Here, the occurrence of such genotypes from these samples is reported and a map of potential risk of emergence of resistance to the main front-line therapies currently used to treat malaria-infected patients and to the next generation of anti-malarial combinations is provided.

The current phase 2b clinical study of artefenomel, an ozonide showing improved pharmacokinetics properties compared to artemisinins, combined with PPQ was designed to assess the efficacy of single oral doses in patients with uncomplicated falciparum malaria in Southeast Asia (Vietnam) and Africa. In addition to the clinical outcome assessment, three molecular markers associated with drug resistance for mapping the potential risks of future treatment failures were investigated in isolates collected before treatment. The frequency of kKelch13 mutations associated with artemisinin resistance, and Pfmdr1 and Pfpm2 genes copy number were measured in available isolates collected from all clinical sites. Artemisinin resistance was confirmed to be still confined in Southeast Asia. A high proportion of kelch13 validated and candidate resistance mutations were observed as well as a new unreported one (C469P) in Vietnamese parasites and the complete absence of these mutants in African isolates. As previously reported [27, 33, 34], a low proportion of kelch13 mutations was detected in African samples and all these mutations have not been shown to be associated to artemisinin resistance [27].

However, a higher proportion (threefold) of parasites with multiple copies of Pfmdr1, a gene encoding a drug efflux pump, was observed in African samples compared to Southeast Asian isolates. This observation contrasts with previous reports showing high frequency of parasites with multiple copies of Pfmdr1 in Asia [35–37] compared to Africa [38–40]. These finding likely reflect the profiles of evolution of P. falciparum populations linked to anti-malarial drug pressure in both continents. Especially, the prevalence of high Pfmdr1 amplification observed in Africa might be linked with the routine use of artemether–lumefantrine as first-line treatment for more than a decade. Indeed, increased pfmdr1 copy number is known to modulate parasite responses to a wide range of drugs including lumefantrine [38, 41, 42]. Supporting this expectation, it seems feasible that such parasites exposed to lumefantrine as monotherapy for several days following clearance of artemether have been selected, while parasites with a single copy have been eliminated. In contrast, the low prevalence of Pfmdr1 multiple copies observed in Southeast Asia could be due to the recent implementation of DHA–PPQ, the removal of the mefloquine drug pressure or both, as the case in Cambodia [18, 20, 43]. Resistance to piperaquine and resistance to mefloquine have been shown to be mutually exclusive in South East Asia. A possible inverse susceptibility mechanism could be at the origin of this observation.

High frequency of isolates with multiple copies of the Pfpm2 has already been reported in recent studies conducted in Cambodia [18, 20, 43]. As the Vietnamese clinical sites (Gai Lai and Binh Phuoc) are located alongside the Cambodian border (Fig. 2), data from this study might reflect an evolving situation where the amplification of Pfpm2 is spreading beyond Cambodia, as described recently [5, 7]. To date, frequencies observed in Vietnamese isolates are not yet as high as the ones observed in Cambodia but might continue to increase in the future.

Unexpectedly, in African isolates, amplification of Pfpm2 gene was shown to occur at a much higher frequency (~ 27% on average across clinical sites in Africa, reaching 30.5% in Burkina Faso and 33.9% in Uganda) than was recently described (from 11.1 to 13.8% in Uganda, 10% in Mali and 1.1% in Mozambique) [28, 29, 44]. Considering the geographical extent and the diversity of the clinical sites in Africa, the high frequency reported at sites distant to each other suggests that amplification of Pfpm2 gene occurred independently in each site. More importantly, since in Southeast Asia most parasites with multiple copies of Pfpm2 also display kelch13 resistance mutations, which is not the case in African samples, it is likely that Pfpm2 amplification originated in Africa, independently of Southeast Asia.

Unfortunately, in vitro or ex vivo drug susceptibility assays were not possible and association between Pfpm2 amplification and clinical resistance to PPQ was not verifiable in the current study. An evaluation is currently ongoing to see whether, and if so to what extent, these markers of artemisinin and PPQ resistance affected the parasite clearance half-life (PCT1/2) and PCR-adjusted 28 days follow-up in patients treated with artefenomel/PPQ (study MMV OZ439 13,003). However, it was recently reported that compared to drug sensitivities measured on Ugandan isolates from 2010 to 2013 (from the same site, namely Tororo), those measured in 2016 to chloroquine, amodiaquine, and PPQ were increased by 7.4, 5.2 and 2.5-fold, respectively [44]. This longitudinal study showed that rather than drug resistance developing to these three anti-malarial drugs, an increase in sensitivity was observed that was correlated with low prevalence of the polymorphisms recently associated with resistance to artemisinins or PPQ. Indeed, clinical resistance to DHA-PPQ has not yet been reported in Africa [45]. Although, the possibility that parasites showing amplification of Pfpm2 observed in the current study are resistant to PPQ without confirmation of in vitro or ex vivo phenotypes cannot be excluded, data reported by Rasmussen et al. [44] suggest that significant occurrence of clinical resistance to PPQ is unlikely. In other words, in Africa it is unclear whether the amplification of Pfpm2 is necessary and/or sufficient for the development of resistance to PPQ. The ongoing analysis relating the markers of resistance to clinical outcome may provide some insights regarding this question. Until very recently, it was still debated whether additional genetic modifications in the P. falciparum chloroquine resistance transporter (Pfcrt) gene are required to confer such resistance [46, 47]. Recent genomic and biological investigations have revealed a rapid increase in the prevalence of novel Pfcrt mutations in Cambodia (H97Y, F145I, M343L, and G353V). These mutants (from culture-adapted Cambodian field isolates or Dd2 gene-edited clones) were confirmed to confer PPQ resistance as determined using the PSA^0–3h [6, 48].

At present, several artemisinin-based combinations are used in Africa and Asia to treat patients with uncomplicated malaria. Artemether–lumefantrine (AL), artesunate–amodiaquine (AS–AQ), artesunate–mefloquine (AS–MQ), artesunate–sulfadoxine–pyrimethamine (AS–SP), dihydroartemisinin–piperaquine (DHA–PPQ) and pyronaridine–artesunate (PA) achieve more than 95% efficacy in clinical trials based on PCR-adjusted Day 28 ACPR. Due to the long post treatment prophylaxis of the well-tolerated PPQ, DHA–PPQ is currently under evaluation in a number of interventions, such as intermittent preventive treatment in pregnant women or in infants (IPTp, IPTi) and mass drug administration campaigns (MDA) in Africa. As a key surveillance goal, it is therefore of particular importance to continue following the evolution of Pfpm2 amplification along with mutations in the Pfcrt gene and to investigate whether these genetic signatures are associated with PPQ resistance in Africa.