ABOVE: IN THE BLOOD: Red blood cells infected with a malaria parasite (purple) circulate with uninfected RBCs (gray).

© ISTOCK.COM, JARUN011

It’s not clear why, but the Greater Mekong Subregion—Cambodia, southern China, Laos, Myanmar, Thailand, and Vietnam—is a major source of malaria drug resistance. Each time a drug has been deployed in the area, resistance mutations in local Plasmodium falciparum, the parasite that causes the mosquito-borne disease, have followed close behind. Parasites there seem more adaptable than P. falciparum in other regions, says Thanat Chookajorn, an assistant professor of biochemistry at Mahidol University in Thailand, who studies the molecular genetics of malaria parasites that thrive in the Greater Mekong.

“It sounds kind of self-centered to say, ‘My parasite’s the worst in the world,’” Chookajorn says. “But I would say that there’s definitely something funny going on with this population.”

Resistance to chloroquine, the first widely used antimalarial drug, first arose in the Greater Mekong shortly after World War II. Chloroquine-resistant strains eventually spread to Africa, which carries more than 90 percent of the global malaria burden. This explosion of drug resistance contributed to an alarming climb in worldwide mortality rates in the second half of the 20th century.

In the 1990s, artemisinin—a compound derived from the wormwood plant that was used for centuries in natural medicine to treat pain and fever—was released globally as a new malaria treatment.1 The drug was a boon to malaria scientists, who were able to pair brief pulses of aggressive, short-acting artemisinin derivatives with longer-acting partner drugs to make artemisinin-based combination therapies (ACTs). These extremely effective treatments—plus intensive programs for implementing rapid diagnostic tests and insecticide-impregnated bed nets—slowed the parasite’s progress. Between 2010 and 2015, global malaria mortality dropped almost 30 percent. Compared to nearly 1 million annual malaria-related deaths in the late 1990s, today only about 400,000 of the 220 million cases per year end in the patient’s death, according to the World Health Organization (WHO).

Starting around 2007, however, ACT resistance slowly began creeping into parasite populations, especially in the Greater Mekong. These days, there is at least one P. falciparum mutant resistant to each ACT partner drug, and a handful have begun to show partial resistance to artemisinin itself, with the drug taking longer to clear the parasites from the bodies of infected people. “This truly is a wily parasite,” says biochemist David Kaslow, vice president of Essential Medicines at the nonprofit global health organization PATH.

Resistance against malaria drugs has been a battle since day one.

With no viable alternative to artemisinin, increasing numbers of malaria infections with delayed clearance following artemisinin treatment are concerning, experts say—and if resistance develops in Africa, the results could be disastrous. Strains with partial artemisinin resistance have drawn a great deal of attention, even being dubbed “super malaria” by some media outlets. “I really caution us against complacency,” says Kaslow. “I don’t think there’s evidence today to say that resistance is going to explode in Africa. But tomorrow, it could. I would not put anything past this parasite.”

Some scientists are less concerned over the danger of ACT resistance, arguing that the threat is overblown. Currently, the ACT arsenal remains strong enough to defeat the parasite, says Sanjeev Krishna, a molecular parasitologist at St. George’s University of London. “If you have the right combination partner, then your treatment is curative.”

Steve Meshnick, a professor of epidemiology at the University of North Carolina at Chapel Hill, agrees that partial artemisinin resistance is, for now, a mere blip in the fight against malaria. “I’m not saying it’s not a problem,” he says, “but I think it gets too much attention.”

ACT of resistance Plasmodium falciparum resistance to artemisinin-based combination therapies (ACTs) started to crop up around 2007. This largely arose from pairing artemisinin derivatives with older drugs that had existing resistance problems. But the emergence of partial resistance to artemisinin itself—which allows parasites to persist for longer in the body following treatment—may also play a role. Partial artemisinin resistance Researchers have linked partial resistance to artemisinin-derived drugs with several mutations in the kelch13 gene, which encodes a binding protein whose role in the parasite’s ability to persist is still unclear. Delayed parasite clearance has also been linked to a prolonged ring stage, which appears to be the only part of the parasite’s lifecycle during which it is able to partially survive artemisinin derivatives such as artemether, artesunate, or dihydroartemisinin. A single dose of these ACT ingredients stays in the body for only a few hours, and patients are typically treated with an artemisinin derivative for only the first couple of day of malaria therapy, so it’s thought that the prolonged ring stage may help the parasites survive the therapy. Preliminary evidence suggests that resistant parasites also rush through the subsequent trophozoite stage, which appears to be the most susceptible to artemisinin.

See full infographic: PDF Data taken from Antimicrob Agents Chemother , 59:3156–67, 2015. Times are approximate.See full infographic: WEB © Tami tolpa 1 Ring stage (most resistant). Parasite has just invaded a red blood cell. 2 Trophozoite (least resistant). Parasite is maturing and getting ready to replicate. 3 Schizont. Parasite replicates and eventually ruptures cell, releasing more invaders and re-starting the cycle.

Malaria’s history of drug resistance

Resistance against malaria drugs has been a battle since day one. Soon after chloroquine’s international release in the late 1940s, parasites began to fight back, particularly in Colombia, Thailand, and Cambodia,2 which were subjected to mass chloroquine treatments, often at low doses that promoted the evolution of resistant parasites. The drug was even added to table salt in some countries in a desperate attempt to curb infections.3

Sure enough, P. falciparum strains in these areas developed multiple mutations in the transmembrane protein PfCRT.4 This allowed the parasite to reduce the accumulation of the drug in its digestive vacuole, where it normally kills the parasite by binding to subunits of oxidized heme, thereby preserving the toxicity of this byproduct of the parasite’s digestion of hemoglobin. Normally, the parasite polymerizes heme subunits into harmless clumps, but the binding of chloroquine prevents this aggregation, preserving the heme’s toxicity. Mutated PfCRT appears to limit chloroquine’s access to the digestive vacuole, allowing resistant parasites to clean up the dangerous scraps of their hemoglobin dinner without interruption. (See illustration.)

Before long, chloroquine was rendered too unreliable to use as a regular treatment against P. falciparum. Researchers developed synthetic alternatives, but these too encountered resistant parasites soon after their release.5 Some drugs also carried risks of severe side effects such as bad skin reactions and liver problems; or, they had complex dosing instructions that reduced compliance, thereby limiting their use as frontline treatments. Mortality rose at an alarming rate, especially in Africa. In Senegal, for example, death rates climbed as much as sixfold in children under 10.6

Enter artemisinin, which would come to be known as a “miracle drug.” Semisynthetic antimalarial formulations of the natural compound, originally developed in China in the 1970s, proved incredibly effective at swiftly killing P. falciparum with minimal side effects. When activated by iron released by the hemoglobin-digesting parasite, the drug is thought to pummel P. falciparum at multiple targets, including ATPases and enzymes important for the redox cycle, the core of fundamental biological processes such as metabolism and cellular respiration. A short, three-day pulse of artemisinin wipes out the vast majority of infecting parasites. When paired with slower-acting partner drugs that mop up any stragglers, artemisinin derivatives such as artemether, artesunate, and dihydroartemisinin (DHA) became unstoppable. By the early 2000s, such ACTs were the go-to malaria treatment. Mortality rates slowed globally, then dropped by the hundreds of thousands.

The problem was that some of the partner drugs had already been around for decades as chloroquine alternatives. That meant various P. falciparum strains had already developed resistance to some of these partner drugs. Because of this, researchers began to see malaria infections resurface after ACT treatment.

The Rise of Malaria Drug Resistance Plasmodium falciparum resistance to artemisinin-based combination therapies (ACTs) started to crop up around 2007. Infections, especially in the Greater Mekong area of Southeast Asia, seemingly survived treatment. This was largely due to the pairing of artemisinin derivatives with older drugs that had existing resistance problems. But some experts think the emergence of partial resistance to artemisinin itself—which allows parasites to persist for longer in the body following treatment—could also play a role. PDF See full infographic: WEB The scientist staff

The rise of ACT resistance

The first widely used ACT paired the existing drug mefloquine, which had been around since the late 1970s, with a blast of artesunate. In the early 1990s, artesunate-mefloquine (ASMQ) was used to treat malaria in provinces on the Cambodia-Thailand border, where existing mefloquine therapies were failing. With an initial dose of artesunate to wipe out the bulk of offending parasites, ASMQ succeeded in curing almost 100 percent of infected patients in the area.7

By the mid-2000s, however, it became clear that artesunate could no longer compensate for the ever-increasing ability of P. falciparum to resist mefloquine. By upregulating the multidrug resistance gene that encodes the mutated transport protein PfMDR1, the parasite likely pumps the drug away from its cytosolic target and into the digestive vacuole to be destroyed. Many parasites that lingered after the artesunate treatment were thus able to survive the mefloquine mop-up. By 2008, ASMQ was failing in about 20 percent of patients in that region of Southeast Asia.8

A new ACT, which combines DHA with the existing drug piperaquine (PP), hit the market in 2007. DHA-PP worked well against ASMQ-resistant parasites, but soon failed against strains from a lineage called PLA1 that somehow resisted PP. Like chloroquine, PP inhibits heme detoxification, but it’s also thought to target plasmepsins 2 and 3, which areproteases the parasite requires to digest hemoglobin for its peptides.9 Without these proteases, P. falciparum starves. By making multiple copy numbers of plasmepsin 2 and 3 genes, PLA1 parasites can overcome the effects of the drug.10 By 2013, 25 percent of patients in western Cambodia weren’t responding to DHA-PP.11

In addition to resistance to the partner drugs in ACTs, P. falciparum has also shown signs of evading artemisinin derivatives. Around the same time that DHA-PP was released, researchers in western Cambodia noticed that it was taking patients longer to clear parasites following the initial pulse of artesunate, DHA, and other artemisinin-derived drugs.12 P. falciparum were found in the blood three or four days after treatment, whereas it normally took just one or two days for the drugs to reduce the infection to below-detectable levels of parasite. Malaria scientists dubbed this phenomenon “partial” or “emerging” artemisinin resistance, because although the treatment was taking longer to work, it was still effective, with most resilient parasites being cleared within a week.13

Investigations into delayed-clearance mechanisms pointed to various mutations in the kelch13 gene, which codes for a poorly understood kinase-binding protein. In vitro studies show that the parasite’s resistance is effective at a specific life stage—the early ring stage, which occurs soon after P. falciparum enters a red blood cell but before it starts replicating. Artemisinin derivatives get metabolized by the body very quickly, so “resistant” parasites appear to evade the drug by lingering longer in the ring stage, biding their time.14 (See "The Rise of Malaria Drug Resistance" illustration.)

I really caution us against complacency. —David Kaslow, PATH

Initially, cases of delayed clearance in Western Cambodia were limited to small, discrete geographic regions where the parasites carried various different kelch13 mutations, none of which appeared to have an advantage over the others, says Roberto Amato, a population geneticist at the Wellcome Sanger Institute. It wasn’t until DHA-PP–resistant PLA1 parasites emerged in 2008 that things started to get bad, he says.

Of all the kelch13 mutants, one haplogroup, called the KEL1 lineage, appeared to have the potential to spread more aggressively than others. When parasites started picking up resistance alleles from both KEL1 and PLA1 together, those KEL1/PLA1 parasites came to dominate the landscape in Cambodia. The so-called co-lineage soon spread to Thailand, Laos, and Vietnam.15 Frequencies of both the PLA1 and KEL1 parent lineages shot up—KEL1, from 4 percent in 2007 to 63 percent in 2013; PLA1, from zero to a whopping 79 percent in that same period. By 2013, more than 90 percent of DHA-PP resistant parasites carried resistance alleles from both lineages.16 “That’s when people really started shouting at each other,” says Amato, with some arguing that ACT resistance could pose serious problems in the fight to eradicate malaria, and others maintaining that the concern was overstated.

By 2017, DHA-PP treatment failure had reached 30 percent in Vietnam and a staggering 90 percent in western Cambodia.17 It’s thought that the KEL1 lineage allowed parasites to persist in patients two or three days longer after the artemisinin pulse than in other endemic areas, while the PLA1 lineage made them resistant to PP. Currently, Amato says, “KEL1/PLA1 has basically replaced the indigenous population [of P. falciparum]. So the situation is not great.”

Counterargument: The problem is resistance to partner drugs, not to artemisinin

Experts caution against sensationalizing partial artemisinin resistance, as there is some debate over the role that it plays in ACT failure. Indeed, there are no known cases in which delayed clearance has led to treatment failure on its own, says Meshnick at the University of North Carolina—resistance to the partner drug, not artemisinin, is the primary driver for an ACT failing. For this reason, he adds, the term “resistance” should not be conflated with delayed clearance. “I think it’s misleading. When you say everybody’s ‘artemisinin resistant.’ . . . All it means is that everybody has delayed clearance times, but they’re still responding to the drug.” If the therapy uses an effective partner drug, it will still be successful—and currently, if one ACT fails, another will still be effective.

It is also reassuring that, so far, P. falciparum’s ability to resist artemisinin treatment is restricted to the ring stage, says Krishna. Ongoing research into the effects of adjusting the artemisinin dose could yield a solution, adds Meshnick. “I’ve heard people talk about giving artemisinin for longer periods of time, or maybe giving it twice a day versus once a day,” he says. “I think all of those things should be on the table.”

Cases involving delayed clearance in Southeast Asia and South America are on the rise, but actual clearance times have not increased further since they first appeared, says Pascal Ringwald, who leads the Drug Efficacy and Response unit for the WHO Global Malaria Program. Thus, the parasites do not appear to be moving toward more complete resistance—at least not yet, he says. “We are concerned, we are watching it, and we are trying to find alternatives. But we can still cure 100 percent of people if we have a good partner drug.”

During the blood stages of malaria infection, the parasite resides within red blood cells, digesting hemoglobin to support its growth and maturation. Partner drug resistance In red blood cells, P. falciparum digests human hemoglobin to feed itself. In addition to amino acids, this releases toxic heme. Normally, the parasite polymer­izes the heme into harmless clumps of hemozoin or degrades it through a handful of poorly understood pathways. But most ACT partner drugs inhibit detoxification. Some partner drugs also attack the parasite through other mechanisms. Here are examples of how P. falciparum strains resist these drugs. PDF See full infographic: WEB © Tami tolpa

Ultimately, most experts agree that ACT resistance needs to be monitored, but when it comes to extinguishing malaria—which has stubbornly maintained a constant global burden since 2015—there are bigger fish to fry. Drug resistance has thankfully not played a role in the current stall, says Pedro Alonso, the director of the WHO Global Malaria Program: it seems ACTs are enough to keep resistance from affecting mortality rates for the time being, including in Africa. Despite occasional reports of artemisinin resistance markers popping up there, none have stuck around, suggesting there is no selective pressure for the parasites to survive the initial blast of the compound’s derivatives. Since the mid-2000s, there have been signs of selection for resistance to lumefantrine, the partner drug of Africa’s frontline ACT, but for now it remains 100 percent effective when combined with a blast of artemether. “There is no drug resistance of any significance in the entire continent,” says Alonso.

Rather, Alonso says, logistical challenges of delivering ACT therapies, diagnostic tools, and other life-saving interventions such as insecticide-treated bed nets have caused declining mortality rates to stall at about 400,000 per year. Furthermore, funding has not increased apace with population growth, meaning that bed nets and on-site clinics are in short supply. “We’re probably seeing the limits of what can be achieved with the tools and the funding that we have.” He and other experts also express greater concern about the evolution of DDT-resistant mosquitoes that could thwart additional efforts to prevent transmission.

Some, however, continue to sound the alarm. Even if delayed clearance doesn’t directly lead to treatment failure, it puts more pressure on partner drugs to succeed in mopping up lingering parasites, says Nicholas White, a professor of tropical medicine at Mahidol University and the University of Oxford. Parasites even developed resistance to lumefantrine, which was never used as a monotherapy before its release in an ACT with artemether in the late 1990s, in the Greater Mekong within just five or six years. And the newest ACT, which pairs artesunate with the partner drug pyronaridine, was already failing in 10 percent of cases in western Cambodia at the time of its release in 2012, and artemisinin clearance times were already two to three days longer.

Even with ample ACT options, changing up the treatments typically given in a particular area is easier said than done, says Amato. For many patients in

difficult-to-access regions of endemic countries with poor access to clinics, simply rolling out rotating partner drugs every time ACT resistance pops up is not always practical. In cases of delayed clearance, researchers are experimenting with tweaking the dose of artemisinin derivative, but this can be equally tricky to implement—for instance, if the derivative can’t be easily separated from its ACT partner because it normally comes in a single pill or blister pack. “There are various strategies on the table,” he says. “The question is, which ones are actually logistically implementable?”

And while the evidence says that partial artemisinin resistance doesn’t cause ACT failure on its own, Alonso acknowledges that, with an ever-evolving parasite, the situation could change tomorrow. “In public health, one learns to never say never.”

The Search for a Better Antimalarial While experts debate the relevance of ACT resistance to the fight against malaria, researchers are always looking for alternatives. One option is to combine existing drugs in new ways. For instance, a Phase 2 clinical trial monitoring more than 2,000 patients throughout the Greater Mekong, southern Asia, and the Democratic Republic of the Congo is investigating whether the partner drug mefloquine can be combined with the ACT dihydroartemisinin-piperaquine (DHA-PP), as the mechanisms by which P. falciparum evades mefloquine and piperaquine appear to be mutually exclusive. Simply recombining existing drugs may not be sufficient, however, and the search for antimalarial drugs with new mechanisms of action—such as damaging the parasite’s digestive vacuole membrane or disrupting its ability to stick to red blood cells—is also underway. A dozen or so drugs, including both alternative partner drugs and novel artemisinin derivatives, are currently in early- to mid-stage clinical development. One new combination therapy, which has shown promise in Phase 2 trials against parasites that exhibit partial resistance to artemisinin, partners the chloroquine-like drug ferroquine with artefenomel, the first potential synthetic alternative to artemisinin. Artefenomel has a much longer half-life than artemisinin-derived drugs, allowing it to be given as a single dose. It has also shown high activity against the resistance-prone ring stage of the parasite. Most of the antimalarials in development attack blood-stage schizonts, the stage that causes illness. However, a handful instead target the gametocyte stage picked up by the mosquito. Such drugs would not cure an existing infection, but could control transmission, a critical aspect of malaria elimination. Two of these, tafenoquine and primaquine, have advanced through Phase 3 trials, but they can’t be used broadly, because they can cause severe damage to red blood cells in patients with a genetic condition called G6PD deficiency that is common in Africa. Another leading candidate, KAF 156, which doesn’t carry the same risk, is currently being tested in Phase 2b trials in combination with the partner drug lumefantrine. With a parasite that continues to evade many existing antimalarial treatments, says David Kaslow, vice president of Essential Medicines at PATH, “we’ve got to continue to invest in new drugs.”

Natalie Slivinski is a freelance science journalist living in Seattle, Washington.

References

Correction (March 5): This story has been updated from its original version to correct David Kaslow’ s position at PATH. Also, while millions of malaria-caused deaths were averted, annual mortality rates dropped by the hundreds of thousands. The Scientist regrets the errors.