The malaria parasite is a wily organism, shifting its life stages as it flits from human to mosquito and back again. It still kills some 600,000 people each year and has outwitted eradication efforts, having developed resistance to previously popular drugs and, thus far, eluded vaccine-induced immunity.

The arrival of a powerful drug in the late-20th century gave researchers new hope. Called artemisinin and based on a traditional Chinese herbal remedy, it cleared the parasite faster and more thoroughly than any other current antimalarial. Researchers are still somewhat uncertain about exactly how it works, but they know that it targets the parasite as it infects red blood cells.

But the hope that artemisinin would serve as a final, exterminanting blow against malaria has begun to fade. Since 2008 patients in Southeast Asia have been slower to lose the malaria parasite Plasmodium falciparum than they once were. And this precursor to resistance seems to be spreading, despite efforts to carefully use artemisinin (by giving it in combination with other drugs) to avoid the emergence of resistance.

Researchers have been tracking that spread—and looking into the genetic basis of the trait—to try to develop more effective ways of keeping the disease in check. The findings, described in two related papers published online April 5 in Science and The Lancet, were called "a tour de force" by David Sullivan, an associate professor at Johns Hopkins Malaria Research Institute, who was not involved in the new research.

Loci of resistance
The confirmation of additional resistance will likely be a blow to malaria battlers. "Artemisinin-based compounds have really been the key to success in pushing back malaria in recent years," says Timothy Anderson, of the Texas Biomedical Research Institute and a co-author on both new studies. "But there's been a cloud on the horizon: patients in western Cambodia have been showing very, very slow parasite clearance." As a result, such patients have a greater chance of having the parasite return. They also provide a wider window for mosquitoes to bite them and then transmit their (more resistant) infection to other people.

For their Lancet study, Anderson and his colleagues (from the U.S. and Southeast Asia) studied the infections of 3,202 malaria patients on the western border of Thailand over the course of a decade. They found that the time it took the artemisinin-based combination therapy to clear the parasites steadily increased, from a mean of 2.6 hours in 2001 to 3.7 hours in 2010. They also found that the infections that persisted with a half-life of more than 6.2 hours jumped from 0.6 percent at the beginning of the study to 20 percent at the end.

These numbers might look small, but they are quickly creeping toward those figures already present in the resistance epicenter of western Cambodia, about 800 kilometers away, where the mean clearance time in 119 different patients was 5.5 hours, and 42 percent of the infections had a half-life of more than 6.2 hours. The researchers predict that the Thailand border area, which has used the artemisinin-based therapy since the mid-1990s, will see these levels of resistance within the next two to six years.

"Slow-clearing, resistant parasites are not restricted to western Cambodia as everyone had hoped," Anderson says. "Our approach to containment really has to be rethought."

The new locus of resistance is also of concern because the western border of Thailand abuts Burma (Myanmar), where malaria is much more common and the public health infrastructure is even less equipped to handle stubborn infections.

New mutations
The researchers also found that the slow-clearing parasites harvested from Thailand were not all that closely related to those in Cambodia, which suggests that rather than having spread geographically, resistance has sprung forth anew.

This observation has researchers even more worried, because it suggests resistance could then emerge elsewhere, such as Africa. "We know from historical examples—from the emergence of other drug resistances—that when resistance does hit Africa, it can spread incredibly rapidly," says Ian Cheeseman, a postdoctoral researcher at Texas Biomed and co-author on the Science study.

History has already shown what this sort of resistance pattern looks like. Former mainstay drugs (chloroquine and sulfadoxine–pyrimethamine) also experienced their first resistance challenges in western Cambodia before spreading to other parts of Asia and on to Africa, where the parasites killed millions. Anderson describes this as a sort of resistance déjà vu.

Researchers also currently lack any quick lab tests to tell if a patient is infected with an artemisinin-resistant parasite. For now, they must simply try the standard treatment and watch and wait to see if it clears in a timely manner.

Nevertheless, Anderson notes, "We've shown that the slow clearance is definitely due to parasite genetic factors—not caused by low-quality drugs, poor nutrition or some other aspect of the patients."

Genetic clues
With new genetic screening technology, researchers have been looking into the DNA of the parasites to see from whence their resistance might be springing.

As Anderson, Cheeseman and their colleagues describe in their Science paper, they analyzed 6,969 spots on the genome of 91 parasites (collected from Cambodia and Thailand as well as Laos, where resistance has not yet been reported). From that data, they found 33 locations on the genome that seem to be under strong selection pressure for resistance, with a hot spot located on chromosome 13. "We've narrowed down the search to a small area of the genome" that confers resistance, Anderson notes.

"This is a huge leap, but it doesn't really nail the mechanism of resistance," Sullivan says. "They've identified several novel areas," he continues, noting, "they still have a handful of genes to look at and really pin down."

Anderson, Cheeseman and their fellow researchers are planning to drill down further into the genetics to root out more specific regions of resistance. "The next step is going to be some fine mapping to define that mutation," Cheeseman says. "By finding out these mutations, we will hopefully be able to know more about this resistance and how this drug works. We don't have a great handle on that yet," he adds.

That more detailed picture should also allow them to better pinpoint just how many individual times artemisinin resistance has emerged already, "and that will allow us to predict the number of times resistance might emerge in the future," he notes.

A list of mutations might also help to point the way to new similar compounds that would still lay the parasite low.

Sullivan notes that although there is a grim history of resistance spreading from this region, unlike previous cases, which have taken more than a decade to characterize, this one has been spotted and described relatively quickly. "If we can identify the gene, then we might be able to circumvent the resistance in some way," he says.

Empty arsenal
Artemisinin has been a linchpin of malaria treatment in recent years, but if it becomes widely ineffective, there is nothing quite ready in the reserves to take its place. "I think the best way to beat microbial resistance is to identify new drug targets," Sullivan says. But, as Anne-Catrin Uhlemann and David Fidock in an essay in the same issue of The Lancet wrote, "drug development efforts are not expected to yield new antimalarials until the end of this decade."

Other drugs in the combination-treatment pyramid have already balked. As Anderson notes, many of the parasites in Thailand were already resistant to another antimalarial, mefloquine, "so the situation was held in the balance by artemisinin."

A malaria vaccine, made by GlaxoSmithKline, is in clinical trials, and a 2011 paper reported promising results, showing that it seemed to protect about half of the kids who received it. But Anderson is not holding his breath and hoping that the vaccine arrives before artemisinin resistance picks up any more speed. "We've been treading water for the past 25 years waiting for the vaccine," he says.

"Drug treatment is still the mainstay of malaria control, and we really don't have another drug to fall back on," Anderson says.