NEW YORK (360Dx) – Improved malaria detection could aid in eradication efforts, potentially saving the lives of a quarter-million children each year. Preliminary data from infected children in Malawi has suggested a breath-based test for malaria is feasible, and the malaria breathalyzer concept will now be validated in a cohort of kids in Kenya using newly-awarded funding from the National Institutes of Health.
More than 200 million people were infected with malaria-causing Plasmodium parasites in 2017 with approximately 90 percent of infections occurring in Africa, according to the World Health Organization. The parasites — which are transmitted by mosquito bites and infect red blood cells — caused an estimated 435,000 deaths in 2017, and children under 5 years old accounted for 266,000 deaths, or about 60 percent of worldwide malaria mortality.
The malaria breathalyzer project is led by Audrey Odom John, a pediatric infectious diseases clinician and researcher Washington University in Saint Louis. The project was awarded funding earlier this month from the National Institutes of Allergy and Infectious Diseases totaling $297,059 over two years.
A breath-based test for malaria could be a real game changer for a few reasons, Odom John said in an interview.
Existing malaria tests — such as rapid diagnostics, molecular tests, or microscopy — rely on blood samples, so they require sterile equipment and people trained to do blood draws. Perhaps as a result, in some parts of Africa as few as 10 percent of kids get any diagnostic testing before they are given anti-malarial drugs, Odom John said.
But a portable diagnostic system that does not rely on blood could increase the proportion of kids with fever who get a diagnostic test, "and that would be huge," she said.
And, in public health scenarios where total eradication of malaria in a community is the goal, a diagnostic that enables testing and treating all the kids in an elementary school, for example, would also be really valuable.
"We know that children are a real reservoir of malaria transmission ... but right now you would have to go in and draw blood from an entire school, and that would not be easy to do," Odom John said. On the other hand, "You could imagine that you could really easily implement [a test-and-treat program] if all you were asking the kids to do was to blow into a bag," she said.
Furthermore, there may soon be a need for new diagnostic options for malaria. Rapid tests, which typically detect a Plasmodium protein in patient blood samples called histidine-rich protein 2, or HRP2, are increasingly failing, and in areas where these tests have been implemented, parasites with deletions of one or more of the genes for HRP2 — namely, pfhrp2 or pfhrp3 —seem to be becoming the dominant population.
An international team of researchers drew attention to this phenomenon in an Emerging Infectious Diseases publication last year, highlighting it as a "major threat to malaria control programs." The study looked at 50 infected patients at two hospitals in Eritrea and showed approximately 60 percent of cases had false-negative HRP2 rapid test results.
In a modeling study published in eLife the authors suggested that HRP2 gene-deleted parasites may soon spread across sub-Saharan Africa, and added that this situation may be one of the first examples of a diagnostic test itself leading to the emergence of resistance. Essentially, performing the HRP2 test has exerted a selective pressure, giving opportunity for a resistant strain to flourish. In support of this theory, the authors of the EID on the phenomenon in Eritrea noted, "clonal expansion of pfhrp2-negative parasites was probably caused by selection by use of HRP2-based RDTs."
The WHO reports that an estimated 276 million rapid diagnostic tests were sold globally in 2017 and three quarters of all testing in sub-Saharan Africa was done using RDTs.
A note from the WHO Global Malaria Program highlighted that most commercially available RDTs use HRP2 as the target, and suggested that regions now consider switching to less-sensitive RDTs that detect plasmodium lactate dehydrogenase, or pLDH, if the prevalence of false-negative HRP2 tests reaches about 5 percent.
"We are about to lose our best diagnostic test," Odom John said, and alternatives will soon be needed.
Assays like microscopy or rapid point-of-care molecular malaria tests from Meridian, QuantuMDx or MolBio might be alternatives to RDTs, although these would require blood samples. A rapid, non-invasive test, on the other hand, could be particularly beneficial in this circumstance.
The insight
The key insight that has enabled the malaria breathalyzer research came from the Odom John lab's initial interest in parasite metabolism. She and her team were investigating metabolic pathways with the goal of developing new treatments, but, she said, they decided to also explore an interesting observation a student made about the cell biology of plants and parasites.
Plants produce volatile organic compounds in their chloroplasts. "These ultimately become the odors and flavors that we associate with plants — it is the way that pine trees smell like pine trees, or roses smell like roses, or lemons smell like lemons," Odom John said.
Many parasites also have a very similar organelle to the chloroplast, called an apicoplast, that isn't used for photosynthesis, but rather for other types of metabolism.
Indeed, parasites with apicoplasts are in the phylum Apicomplexa, with disease-causing phyla members including Plasmodium but also parasites like Babesia, Cryptosporidium, Cyclospora, and Toxoplasma gondii.
The researchers had the "kind of crazy idea," Odom John said, that if the apicoplast were so similar to the chloroplast, a relic of some ancient symbiosis with chloroplast-containing algae, perhaps the apicoplast may also produce VOCs, which could in turn be excreted by the human host and act as odorants to lure mosquitoes.
The group cultured Anopheles gambiae plasmodium parasites in human red blood cells and set up solid-phase microextraction fibers above the culture dish, which can selectively bind any nonpolar organic compounds floating in the air.
And indeed, as described in mBio in 2015, they found that with a large enough culture volume the plasmodium parasites produced detectable levels of gasses.
Interestingly, it seems the parasites even mimic the plants that their mosquito hosts prefer. Mosquitoes subsist on nectar, only consuming a blood meal as part of their reproductive process, and the odorants the parasites produced corresponded to some that are made by the plants Anopheles gambiae tend to prefer, such as flowers in the aster family – like daisies, dahlias or dandelions – as well as castor bean plants. Generally, the compounds were from a class of VOCs called terpenes.
Subsequent work was done by the team on naturally infected children in Lilongwe, Malawi. As described in the Journal of Infectious Diseases last year, that work demonstrated that numerous compounds are detectable upon "breathprinting" children who are infected with Plasmodium falciparum.
A specific panel of six molecules in breath was able to classify infections with an 83 percent accuracy, and two of these compounds were terpenes — α-pinene and 3-carene, specifically — that were also found in the mBio experiments to be lingering in the air above cultured Plasmodium.
Children are the ideal patients for whom to develop a malaria test, Odom John said, in part because they typically carry much higher numbers of parasites when they are infected than do adults. "It would be hard to imagine that if you can't find it in children, you could find it in adults," she said. And, as a primary reservoir of infection in communities, knowing the biomarkers of infection in children might lead to a diagnostic with the most impact.
Now, the group will use the new funds to replicate the Malawi study in Kenya. They will also try to understand at what stage in the parasite's life cycle it produces the VOCs. More research is needed, but Odom John hopes that eventually the piney-smelling substances might be detected from children in the field using a point-of-care instrument.
The growing field of breath-based diagnostics
"The idea of diagnosing disease based on smell dates back to Hippocrates," Odom John said. She noted that the International Association of Breath Research hosts an annual conference, called the Breath Summit, and "there is a growing sense of breath research as a field unto itself," she said.
Increased collaboration is important, because some the breath-based biomarkers may be unique to a particular disease while others could reflect an underlying biological process which may be similar between infections or diseases, she said.
In terms of an actual detection device, the samples collected in Africa are currently shipped back to the US for analysis in the lab. But the Department of Defense has been funding bioengineering research into sensor arrays for things like bomb sniffing, Odom John said, and this might someday translate to breath testing.
"There is a lot of interest and development towards the scientific problem of detecting a small amount of a specific compound from a complex mixture," she said, suggesting that this engineering problem may be solved in the next five years. "Then, it's just a matter of tuning those arrays to detect the specific compounds of interest," she added.
A review of the use of VOCs for detection of infectious diseases also highlighted that a number of bacterial pathogens may also be amenable to breath testing — for example M. tuberculosis or Pseudomonas aeruginosa — and the authors suggest these could be detected using "e-noses," such as the Cyranose from Sensigent.
Other groups are currently attempting to commercialize various applications of breath-based testing.
Owlstone Medical is developing a breath-based biopsy test for lung and colorectal cancer, as well as a breathalyzer for treatment selection in asthma, and recently signed a deal with Astra Zeneca to develop breath-based biomarkers.
Avisa Pharma, meanwhile, is developing tests that require patients to inhale a compound first, and targeting pneumonia and Clostridium difficile infections as initial applications. Exalenz Bioscience is using a similar technology for H. pylori detection. Beyond infectious disease or cancer, Israeli researchers recently reported a breath-based assay that detected early stage Parkinson's disease using a sensor array in a small sample of patients.
And, a group at the London School of Hygiene and Tropical Medicine recently described another set of odorants that are released from the skin of malaria-infected people and attract mosquitoes. In those experiments, aldehydes were detected in body odor of infected people, although that research did not detect the same terpenes in skin that Odom John's team has found in breath.
The London group has been experimenting with training sniffer dogs to detect malaria parasite-induced skin odors, with proof-of-concept data presented last year at the American Society of Tropical Medicine and Hygiene annual meeting.