Since 2010, a biological drama has been playing out in the bloodstreams of humans from Peru to Ethiopia. Plasmodium falciparum, the parasite that causes the most common form of malaria, has managed to evolve away from the tests humans created to track it.
These tests look a bit like the rapid antigen tests that have been deployed during the COVID-19 pandemic. “You put a little blood sample on them, and you get one or two lines,” explains parasitologist Audrey John. The tests are especially valuable in areas where lots of people come to hospitals or clinics with fevers, but only some of them have malaria. They make it possible for doctors to easily recognize malaria patients, so that only those who need medication receive it—and quickly.
But there’s a problem: These tests work by detecting the presence of histidine-rich protein 2 (HRP2), a substance that the malaria parasite makes and disgorges as part of its metabolic process. Unfortunately, that protein isn’t strictly necessary for the parasite’s replication or transmission. The consequence is that, by helping doctors zero in on that protein, the tests inadvertently act as an evolutionary pressure. Any parasites that, by random mutation, don’t emit HRP2 pass under the radar. Their hosts are less likely to receive treatment, and the parasites live to fight another day.
About a decade ago, scientists started to identify cases of malaria in Peru, then in India and East Africa, involving parasites that didn’t produce HRP2 and therefore generated false negatives on malaria tests. According to the World Health Organization, of the 228 million cases of malaria in 2018, 93% were in Africa, and P. falciparum caused 99.7% of those infections, making the appearance of the mutation a big deal.
The trend is strongest in places with comparatively few malaria cases and where sick people are likely to get treatment, John says. A review of world malaria studies found that the mutant parasites had been reported from all major malaria-endemic areas. And while there are gaps in the data, analysis of multiple studies suggests that in parts of Eritrea, Ghana, Sudan, Nicaragua, and Peru, at least 20% of malaria cases may be caused by parasites that no longer make the protein, with numbers higher than 80% in some pockets.
A 2017 study modeling the spread of the mutation in sub-Saharan Africa suggested that in many areas HRP2 tests will no longer be useful by 2030. “This is a total ‘Jurassic Park’ story,” John says. “Life finds a way.” In response, she and her team are working on a novel solution: a breathalyzer for malaria.
Healthy human breath contains some 1,900 volatile organic compounds (VOCs). Generally speaking, VOCs are carbon-based molecules that drift around in the air, often shed from surfaces. They are what our noses pick up on when we smell a scent. The VOCs in human breath are emitted by the body as it functions—or by the menagerie of bacteria that call that body home—and become airborne when they rocket out of our lungs along with exhaled carbon dioxide. The membranes in our lung alveoli are extraordinarily thin, which allows oxygen in and carbon dioxide out of the bloodstream, and that provides an opportunity for these VOCs to escape as well. It is perhaps unsurprising that exhaled breath can include VOCs produced by (or in response to) the microbes that cause respiratory infections like tuberculosis and influenza, which burrow into the tissue around the alveoli. But studies also indicate that breath can include VOCs originating from microbes in other parts of the body, as those waste compounds make their way into the bloodstream and pass through the lungs.
Since 2015, John and several colleagues have been running studies in Malawi and Kenya searching the breath of children with malaria for a so-called “breathprint” of the disease, a pattern of VOCs that can serve as a telltale sign that parasites are at work and the body is responding. They identified six compounds, four of which often form part of human breath but appear to shift in concentration in the presence of malaria. Hunting for those VOCs in specific combinations and concentrations, they were able to successfully diagnose malaria with 83% accuracy. (Remarkably, the other two VOCs are “terpenes,” a compound produced by P. falciparum. Terpenes are similar to chemicals given off by plants that the parasites’ mosquito hosts like to visit for nectar. That means the parasites are hijacking their human hosts’ biology to manufacture a chemical invitation to the mosquito that ferries the parasite from one human to another.)
With the breathprint biomarkers established, the next challenge is designing a breathalyzer component known as an “electronic nose” to sniff out this complicated signature. “A breathalyzer for alcohol is really only trying to detect one thing—one thing that’s very rarely in your breath unless you’re drinking,” John says. It’s much more difficult to detect specific shifts in the concentration of multiple compounds, some of which are already there to begin with.
Still, John remains optimistic, in part because the COVID-19 pandemic has given the project extra momentum. Since she and her team already had a pipeline in place for a disease breathalyzer, it made sense to apply that principle for COVID, she says. Working with children coming to their emergency room, John and her colleagues at Children’s Hospital of Philadelphia set up a pilot study to find a breathprint for juvenile COVID-19. They ultimately settled on a set of biomarkers that allowed them to detect the virus with close to 90% accuracy.
Both a COVID breathalyzer and its malaria-detecting cousin would contain material that can bind to the telltale sets of VOCs and change the resistance of a circuit, plus a little computer that can read the activity on that circuit. With that concept in place and the data in the process of validation, the next step is trying out a trial device in a real-world setting and comparing it to tests whose performance we already trust, John says. She also hopes to continue investigating new breathprints, starting with Plasmodium vivax malaria (the second most common type) and asymptomatic malaria, which is notoriously difficult to diagnose.
All that requires more funding. But John hopes the technology needed for these tests could develop in tandem, with each project building on the other’s successes. With the pandemic raging on, “a lot more people are interested in screening for COVID than treating malaria,” she says. But all over the world, in the bloodstream of millions, life continues to find a way.