December 3, 2024 | Most people in the United States have gotten no closer to malaria than the evening news, making the devasting nature of the infection in sub-Saharan Africa hard to fathom. Possibly once or more a day, people are being bitten by pathogen-carrying mosquitoes that cause untold hours of missed work and school and ultimately claim the lives of over half a million people—most of them young children.
“There is no disease like it where exposure to an infection is so great,” according to Sunil Parikh, M.D., an epidemiologist at the Yale School of Public Health. It is also one of the oldest known diseases. Malaria has been around for thousands of years, with the causal parasites evolving alongside their human hosts.
Up until the last decade or two, when rapid diagnostic testing for malaria infection became available, the gold-standard detection method for the prior 100 years was to examine a blood sample under a microscope. But microscopy has several drawbacks when deployed in the field, including most notably the need for electricity, a working microscope, and skilled microscopists who can identify parasitic pathogens. If present in very small numbers, an entire slide may at best turn up a single parasite, he notes.
While the newer antigen tests are fast, delivering results in 15 to 20 minutes, they likewise have several disadvantages that limit their utility in detecting the disease. This means many people aren’t being reliably flagged for treatment with malaria drugs that could reduce the burden of illness and death, says Parikh.
As with a traditional blood smear test, a rapid finger prick assay is still an invasive test and that can itself be an impediment, depending on how often somebody is being tested, he adds. It also utilizes a small volume of blood making it unable to detect levels of infection below approximately 100 parasites per microliter of blood. “The majority of people walking around in the world probably have a level below that,” Parikh points out, including individuals who are infected but asymptomatic or have developed some level of immunity.
Additionally, some emerging strains of these parasites no longer produce the antigen used in most of those rapid tests, continues Parikh. Rapid tests also cannot quantify the number of parasitized red blood cells in a sample. “It’s just a yes/no test.” When levels of infection are high, infections can be fatal, so knowing the level of infection is important.
Many of these limitations may be overcome with a new noninvasive malaria test under investigation that looks for evidence of the parasite in red blood cells in circulation, allowing almost 200 microliters of blood to be tested in a matter of 10 seconds, and even more if testing over several minutes, Parikh says. The Cytophone, a portable photoacoustic flow cytometer platform, was recently validated in a first-in-human study that was published in Nature Communications (DOI: 10.1038/s41467-024-53243-z).
In tests with 20 adult patients diagnosed with symptomatic malaria in Cameroon, the Cytophone was able to detect malaria infections with 90% sensitivity and 69% specificity. The device performed as well as microcopy and rapid diagnostics when compared to one another and to quantitative PCR, but Parikh says he expects the Cytophone will ultimately outperform PCR due to the ongoing work of bioengineers in the lab of Vladimir P. Zharov at the University of Arkansas, who developed this technology.
“They have already generated improvements to the first-generation prototype with a more advanced laser setup, improved processing and better [ultrasound] transducers,” says Parikh, adding that it has done well in follow-up testing in Cameroon. His confidence in the Cytophone stems primarily from the platform’s ability to interrogate a larger volume of blood, he says. “All diagnostics are really limited by sample volume.”
As a young boy, Parikh made many trips to India where his extended family lives and thus saw firsthand the ravages of malaria in that part of the world. During his formative years, he also traveled to Kenya and Tanzania, where the toll of malaria infections and deaths are hard to comprehend, he says.
As a medical student, Parikh worked on a project in the Amazon region of Peru where both malnutrition and malaria are prevalent. He was then introduced to “the lab side of things” during his residency. With each experience up to this day, he says, he has come to better appreciate the incredible complexity of tackling the disease from the standpoints of science and public health—and the “immense power of multidisciplinary collaboration.”
It was only in working across the scientific disciplines of physics, engineering, clinical medicine, and parasitology that the Cytophone came to be tested as a malaria detection device in Cameroon, says Parikh.
The capacity and ability of colleagues in Africa to conduct clinical studies is noteworthy, he says, speaking from his 20-plus years of doing collaborative research in three different countries on the continent. This has been no less the case in Cameroon where the partnership is more recent, highlighted by the fact that the latest malaria study launched in the early days of the COVID pandemic and investigators there ably took the reins when their teammates were forced to fly back home. “Without our amazing colleagues in Cameroon who live and breathe this disease on a regular basis, our study would not have been possible.”
The bioengineering team in the Zharov lab has been developing the Cytophone technology for well over a decade now, Parikh reports. The technique combines light, soundwaves, and cytometry—an established laboratory technique for measuring the characteristics of cells.
The Cytophone was initially developed to look for circulating melanoma cells in patients with metastatic skin cancer, and the team in Arkansas has made great progress on that front, says Parikh. Melanoma cancer cells have a unique property whereby if you expose them to lasers at a certain wavelength, they will absorb that laser energy more than a normal cell would.
Malaria parasites that infect red blood cells chew up hemoglobin, and in the process release iron that—as a matter of survival—they package into large crystals within the cell, he explains. That leads to properties that make it possible to leverage the photoacoustic flow cytometer platform for malaria detection. “If you expose infected cells that have these iron crystals to lasers, they will absorb more of the light and energy from that laser [than non-infected cells] when used at specific safe wavelengths.”
When that laser energy is absorbed, it creates warmth within the cell that in turn generates sound waves that are detectable by ultrasound, says Parikh. A small probe gets placed on the back of a person’s hand, focused on a blood vessel, and delivers a series of laser pulses. The sound waves created by infected cells as they come by get picked up and software processes those signals.
In the end, the Cytophone produces a trace of that activity where the photoacoustic peaks of the infected cells are highlighted, Parikh says. The platform features a bunch of ultrasound transducers in an array to capture the full cross-section of a blood vessel, since cells aren’t flowing by single file, thus increasing the likelihood that infected cells will get detected. It also accommodates interference that can come from darker skin pigmentation.
The existence of malaria pigment crystals is one of the oldest pieces of knowledge about the disease in recorded literature, says Parikh. The dark “hemozoin” crystals are often easily visible with even a regular light microscope.
Drugs have come to market interfering with the pathway that creates these crystals, he adds, including the longstanding malaria treatment chloroquine. All species of malaria parasites that infect humans produce hemozoin and need to produce it to survive within the cell, meaning none of them are going to figure out a way to stop making it. “It is an essential target that can’t ever not be present in an infection.”
As Parikh sees it, there are multiple ways that he and his colleagues could move forward with the malaria detection platform. “You can envision this being used for screening in places where there is not a lot of malaria, and you don’t want to be getting a blood sample on everyone who is walking through the door; that would not be very cost-effective or palatable to people. But you can also envision [its utility] in a more of a high-volume setting where there is a lot of malaria, and you don’t want to be doing blood sampling on a 100 people in a day, especially in young children.”
If the research team can find a way to quantify the level of malaria in patients using the Cytophone-based device, which Parikh believes is possible, then it might well be useful in identifying patients who are at a high risk of severe disease. Additionally, the test may also be useful for monitoring patient response to malaria drugs. “Every drug ever used for malaria has succumbed to resistance,” he notes. “It is inevitable, and this [platform] could be used to identify people that are responding to treatment slower than would be expected, which may be a sign of resistance.”
The two remaining questions that the research team plans to investigate are ways to quantify how many malaria parasites are present in a sample as well as identify the species. While more than 90% of the parasites that are present in Africa are Plasmodium falciparum, the deadliest variety, the second most common malaria parasite found in South America and parts of Asia is Plasmodium vivax and requires a different drug to get rid of completely, Parikh says.
“The drugs we use to treat people when they’re sick can be used for all types of malaria,” he adds. But for people sickened by P. vivax, the parasite can reawaken after lying dormant in the liver for weeks if not years.
A second-generation Cytophone has already been developed by the Zharov lab and was recently used in a study in asymptomatic patients to determine the lowest detection limit of the device, says Parikh. The device itself is about the size of a larger countertop printer, but the team in Arkansas is intent on miniaturizing the unit once all its desired performance characteristics (few false negatives and false positives) have been demonstrated.