Artemisinin remains the most important antimalarial drug despite the development of partial drug resistant Plasmodium falciparum parasites in Southeast Asia (Dondorp et al., 2009; Ashley et al., 2014), and emerging partial resistance in East-Africa (Uwimana et al., 2020; Balikagala et al., 2021; Uwimana et al., 2021). It is the fastest acting antimalarial drug available in the market, and plays a key role in the treatment of uncomplicated and severe malaria. Artemisinin-based combination therapy (ACT), given orally for 3 days, is the first-line therapy recommended in the treatment of uncomplicated P. falciparum malaria. Parenteral artesunate is the first line treatment for severe malaria, and should be continued until the patient is well enough to receive oral medication. While the world is aiming to eliminate malaria, artemisinin resistance threatens this objective. This review aims to describe the pharmacological properties of ACTs, the mechanism of artemisinin resistance and the potential changes needed in the treatment regimens to fight resistance.

Artemisinin is a short acting but very potent antimalarial drug. It has a short terminal elimination half-life of approximately 1–3 h, resulting in undetectable drug levels within 10 h of drug administration (Tarning et al., 2012; Kloprogge et al., 2015). This pharmacokinetic property demands daily dosing to achieve effective treatment. However, a relatively high degree of recrudescence was seen with short courses of daily monotherapy (<7 days), due to artemisinin-induced dormancy in P. falciparum infections (Peatey et al., 2021). Artemisinin is no longer recommended to be used as monotherapy, except for parenteral treatment of severe malaria. In ACTs, artemisinin is responsible for eliminating the majority of the parasite biomass during the first days of treatment, while residual partner drug concentrations kill the remaining parasites to prevent recrudescence (Figure 1). The partner drug concentrations must be maintained above the minimal inhibitory concentration (MIC) until the infection has been cleared for successful chemotherapy. If the drug concentrations fall below the MIC value before the infection is cleared, parasites could multiply and result in a recrudescent infection (Figure 1).

This time period, after drug administration, where residual drug concentrations are maintained above the MIC value is also referred to as post-treatment prophylaxis. Novel infections emerging from the liver during this time-period will be eliminated and therefore prevent reinfections to be established. The suppression of novel infections associated with long-lasting ACTs are utilized during the prevention of malaria (i.e., prophylactic treatment, seasonal malaria chemotherapy (SMC), intermittent preventive treatment (IPT)). The duration of post-treatment prophylactic effect depends on both the terminal elimination half-life of the drug and the susceptibility of the parasite to the drug, which determines the MIC. ACTs containing a partner drug with a long terminal elimination half-life can be used effectively in the prevention of malaria. The most promising combination is dihydroartemisinin-piperaquine, due to the long biological half-life of piperaquine (20–30 days), which allows monthly treatment to prevent malaria (Hoglund et al., 2017; Chotsiri et al., 2019).

Parasite drug resistance is the result of genetic changes that confers reduced drug susceptibility. This allows a mutated parasite to multiply when exposed to otherwise suppressive drug concentrations. Such a survival advantage, compared to wildtype parasites, would quickly be selected for during high drug pressure in a region, allowing the drug resistant strain to spread. Genetic mutations are by definition random events, and the absolute number of genetic mutations during an infection is therefore expected to increase with increasing parasitemia. Thus, de novo drug resistance is more likely to arise in patients with high parasitemia. The probability of drug resistance development would therefore be higher in patients with hyperparasitemia (>10¹² parasites), compared to patients with uncomplicated acute symptomatic infections (10⁸–10¹² parasites), compared to patients with asymptomatic infections (<10⁸ parasites). When/if such an event occurs, exposure to another drug with a different mechanism of action would eliminate the mutated parasite and prevent the drug resistant parasite to establish a patent infection. This is the main reason why combination therapies prevent drug resistance development. The ideal combination of drugs would be two (or more) highly potent compounds with different mechanisms of action, and matching pharmacokinetic properties (i.e., similar terminal elimination half-lives). This would allow drug concentrations to be maintained above the MIC value for a similar duration of time in order to prevent emerging resistant parasites to establish a patent infection.

The artemisinin class of drugs (i.e., artesunate, artemether, and dihydroartemisinin) is the most potent and rapidly acting antimalarials on the market. Artemisinin commonly result in approximately 10³–10⁴-fold reduction in parasitemia per parasite life-cycle of 48 h, while the marketed ACT partner drug is less potent, resulting in approximately 10¹–10³-fold reduction in parasitemia per parasite life-cycle of 48 h. It would be highly advantageous with novel partner drugs that are more potent than the currently available antimalarials. This rapid reduction in parasitemia associated with ACT reduces the likelihood of resistance development. However, decades of preceding artemisinin-based monotherapy resulted in the development of artemisinin-resistant parasites in Cambodia, which is now widespread throughout mainland Southeast Asia, resulting in a slower parasite clearance of P. falciparum Kelch13-mutated parasites compared to wildtype parasites (Ariey et al., 2014; Ashley et al., 2014; Kagoro et al., 2022; Zhu et al., 2022). Artemisinin resistance has now also emerged in India (Chakrabarti et al., 2019; Das et al., 2019), in South America (Mathieu et al., 2021), and on the African continent in Uganda and Rwanda (Uwimana et al., 2020; Balikagala et al., 2021; Uwimana et al., 2021). Artemisinin-resistant infections are defined as patients showing a parasite clearance half-life above 5 h, at standard dosing (i.e., <10³-fold reduction in parasitemia per life-cycle) (Dondorp et al., 2009). This results in a substantially higher parasite biomass to be eliminated by the partner drug in ACTs, and as a consequence, multi-drug resistant parasites have now developed in Southeast Asia with reduced drug susceptibility towards both artemisinin and its partner drug (Amaratunga et al., 2016). We and others have suggested to use longer duration ACT courses and triple ACTs (TACTs) in the fight against multi-drug resistant malaria. The additional third drug would be selected to match the pharmacokinetic properties of the existing ACT partner drug (e.g., dihydroartemisinin-piperaquine + mefloquine). TACTs have shown excellent treatment efficacy in regions of multi-drug resistant malaria, and they are likely to protect against further drug resistance development (van der Pluijm et al., 2020; Peto et al., 2022).

ACT-related elimination of a malaria infection results in an exponential decay of parasites. The time it takes to clear an infection is dependent on two variables (1) starting parasitemia and (2) rate of decline (Figure 2). Thus, it takes a longer time to clear an infection with high admission parasitemia (hyperparasitemia) compared to an infection with low admission parasitemia (asymptomatic). Similarly, it takes a longer time to clear resistant infections (slower rate of decline) compared to drug susceptible infections (higher rate of decline). Therefore, it is substantially higher risk for resistant development and more difficult to treat symptomatic patients compared with asymptomatic patients. Increased drug pressure in malaria elimination campaigns, resulting from mass-drug-administrations or screen-and-treat strategies, might not increase the risk of resistance development, when using high-quality drugs administered according to standard guidelines. Most individuals receiving these treatments would be asymptomatic patients, with very low levels of parasitemia. Their infections could be treated relatively quick. The low starting parasitemia and the relatively short duration of infection, would make these individuals far less likely to develop antimalarial drug resistance.

Artemisinin resistance was first reported in Cambodia (Dondorp et al., 2009; Ashley et al., 2014), and have now spread throughout the Greater Mekong Subregion (GMS) (Imwong et al., 2020). Clinical artemisinin resistance can be detected by several in vitro susceptibility tests, i.e., the trophozoite inhibition maturation assay (TMI) with an IC₅₀ value >5 ng/ml (Chotivanich et al., 2014); and the ring survival assay (RSA) with a parasite survival rate ≥1% (Witkowski et al., 2013a; Witkowski et al., 2013b; Davis et al., 2020). Molecular studies have demonstrated that mutations in the P. falciparum Kelch 13 (PfK13) gene are associated with a prolonged clinical parasite clearance (Ariey et al., 2014). The PfK13 protein is involved in the endocytosis of hemoglobin, which is required for parasite growth in red cells. The strong association between PfK13 mutant alleles, in vitro parasite survival rate (TMI and RSA), and in vivo parasite clearance half-life indicate that PfK13 mutations are an important molecular marker of artemisinin resistance (Wang et al., 2015; Thuy-Nhien et al., 2017; Sá et al., 2018; Chhibber-Goel and Sharma, 2019; Das et al., 2019; Zhang et al., 2019; Miotto et al., 2020; Siddiqui et al., 2020). PfK13 contains six β-propeller Kelch domains and mutations in this region, particularly the C580Y mutation, has been used in the surveillance of artemisinin resistance in the GMS. However, artemisinin resistance (i.e., prolonged parasites clearance half-life) has also been reported without PfK13 mutations in clinical isolates from Cambodia (Mukherjee et al., 2017) and India (Das et al., 2021).

The following mechanisms have been proposed to explain the development of artemisinin resistance; 1) reduced ring stage drug susceptible compared to the mature stages (Wang et al., 2018; Intharabut et al., 2019), 2) enhanced adaptive responses against oxidative stress and protein damage (Mok et al., 2015), 3) lower levels of ubiquitinated proteins, resulting in enhanced cell stress response and delayed cell death (Dogovski et al., 2015; Tilley et al., 2016), 4) an elevation of a lipid phosphatidylinositol-3-phosphate (PI3P) as a result of the reduced binding between P. falciparum phosphatidylinositol-3-kinase (PfPI3K) and mutated PfK13 (Mbengue et al., 2015; Suresh and Haldar, 2018), 5) other unknown mechanisms protecting the infected red cell from proteopathy (Rocamora et al., 2018), and 6) better adherence to host receptors and remodeling of the infected red cell membrane by vesicle amplification to avoid splenic removal (Bhattacharjee et al., 2018).

Dormant P. falciparum parasites after artemisinin exposure have been reported in vitro (Teuscher et al., 2010) and in vivo (Peatey et al., 2021). This has raised the concern over the potential association of the artemisinin-induced dormant parasites and the development of drug resistance as found in other microbes (Lewis, 2007). Besides, a study on P. falciparum infection in the Aotus monkey model showed little effect of PfK13 mutations in the recrudescent frequency after artemisinin treatment (Sa et a., 2018). This could be a result of dormant parasites or the differences between humans and monkeys in host-parasite relationships. The possibility of artemisinin-induced dormancy emphasizes the importance of partner drugs in ACTs.

Immunity is also suggested to play important roles in the emergence and spreading of antimalarial resistance. Non-immune people almost always become ill when they are first infected with malaria. Therefore, young children in endemic areas are the main group that suffers from the infection; many develop severe malaria and die. With repeated exposure, people can acquire immunity against malaria although the immunity is slowly developed and wanes after a few years if not constantly exposed to the infection. This naturally acquired antibody-mediated immunity can lower parasitemia due to its effect on blood-stage parasites (Marsh and Kinyanjui, 2006), and reduce transmission due to its effect on gametocytes and sporozoites (John et al., 2005 and John et al., 2008; Bousema and Drakeley, 2011). Therefore, the risk of severe infection and death are lower in older children and adults in malaria endemic areas. Interestingly, it has been noticed that although antimalarial resistance can be found worldwide, the origin of resistance is typically in low transmission areas. Chloroquine resistance in Plasmodium falciparum appeared in Thailand (Harinasuta et al., 1962) just 2 years before Colombia (Moore and Lanier, 1961) during late 1950s, both of which were low transmission areas. The resistance continuously spread across Southeast Asia to India and arrived Africa in 1978 (Campbell et al., 1979; Fogh et al., 1979). This route of spreading the resistant genotype was later confirmed by genetic microsatellite analysis (Wootton et al., 2002). Decades later, the history has repeated itself with sulfadoxine-pyrimethamine resistance (Roper et al., 2004) and now with artemisinin resistance.

Suggested explanations of this phenomenon have been proposed recently (Ataide et al., 2017; Whitlock et al., 2021). A multinational study of artemisinin resistance in Southeast Asia demonstrated that faster parasite clearance time was associated with higher immunity levels, suggesting that patients with low immunity are more likely to develop, harbor and transmit mutant parasites (Ataide et al., 2017). A multiscale, agent-based model by Whitlock et al. also provided supportive findings, showing that high host immunity, as acquired in high transmission setting, slowed the evolution of resistance. However, once the resistance becomes common in the parasite population, it will be maintained at a high prevalence (Whitlock et al., 2021).

With global malaria elimination programs, malaria transmission and thus host immunity will be lower in many areas, resulting in greater risk of emergence and expansion of artemisinin-resistant P. falciparum infections.

Multi-drug resistant parasites have now emerged as a consequence of widespread artemisinin resistance, resulting in high failure rates of the ACT dihydroartemisinin-piperaquine in western Cambodia and nearby regions such as northeast Thailand and southern Vietnam (Amato et al., 2017; Witkowski et al., 2017; van der Pluijm et al., 2019). In India, longitudinal Plasmodium falciparum isolates from across the country showed decreased in vitro artemisinin-sensitivity as early as 2012 (Chakrabarti et al., 2019) and declining clinical efficacy of artesunate-sulfadoxine-pyrimethamine has been reported (Mishra et al., 2014; Das et al., 2021). Artemisinin is the most important antimalarial drug and remains a critical tool for malaria elimination and eradication programs worldwide. It is crucial to understand where resistance occurs geographically and the different resistance mechanisms and parasite-host interactions associated with reduced drug susceptibility. Clinical studies and epidemiological surveillance are needed for the containment of artemisinin resistance. Identifying the clinical efficacy of available drugs and combinations, and developing new antimalarial compounds with novel mechanisms of action is needed urgently.

Although resistance to artemisinin and its derivatives has emerged among P. falciparum in various regions (Ashley et al., 2014), ACTs are still the main stay of falciparum malaria treatment. Several researchers have explored various ways to improve the use of artemisinin to maximize its benefits. Modification of existing antimalarial regimens has been considered as the quickest way to improve the treatment outcome. Potential modifications include a prolonged ACT course, alternate use of different ACT regimens, and triple ACTs. Combining a malaria vaccine and an ACT in mass drug administration (MDA) campaigns in low transmission settings have been suggested to accelerate the elimination of falciparum malaria.

This review provides an overview of artemisinin resistance, focusing both on clinical and laboratory aspects. The current efficacy of artemisinin-based combination treatments in areas of multi-drug resistant malaria and the widespread emergence of artemisinin resistance, emphasize the importance of global malaria eradication/elimination programs. In-depth understanding of the pharmacological properties of antimalarials and resistant mechanisms is crucial in the use of modified ACTs in the fight against malaria. The potential modifications included three methods to improve the efficacy; 1) prolonged ACT treatment course, which is easy to apply with existing regimens but concerns have been raised on possible side effects of the long half-life drugs and patient compliance; 2) alternate sequential use of different ACT regimens in a region, which can also be applied with existing regimens but additional information are needed on optimal drug rotation timing; and 3) triple ACTs, which has additional benefits over the first two methods of increased efficacy and the ability to delay the development of drug resistance but drug-drug interaction data are needed to support the global implementation of TACTs. Another potential use of ACTs is in combination with malaria vaccine in MDA efforts to accelerate the elimination of malaria in low transmission setting.