Hello and welcome to the Johns Hopkins Malaria Minute Extended, a podcast sharing the human stories behind the world’s leading malaria science.

Today, an innovative, non-invasive diagnostic tool that could revolutionize malaria testing, with the potential to be built into wearable devices. With Sunil Parikh, Vladimir Zharov and Yap Boum.

Malaria tests are essential to disease control. They’re the first step in understanding a patient’s symptoms and ensuring they get the right treatment – so they need to be accurate and sensitive.

The two most common tests are rapid diagnostic tests and lab microscopy. Rapid tests, like a COVID test, detect key antigens to give a visual indication of positive infection, whereas lab microscopy involves a trained expert looking under the microscope for the parasites themselves.

Both tests require a blood draw. 

Here’s Professor Sunil Parikh, Professor of Epidemiology and Infectious Diseases at Yale School of Public Health.

Sunil Parikh: Both of these point-of-care tests require a blood sample, so most of the time it’s through a finger prick with a small amount of capillary blood. So we’re talking anywhere from 5 to 10 microliters of blood. So they’re invasive.

However, alongside being invasive, you have the small volume used for both of these tests. And because of that you also have limitations in sensitivity for both tests. This is a little bit of an oversimplification, but you need about 100 parasites per microliter of blood before these tests become positive or are able to identify an infection. I think overwhelmingly the reservoir of malaria infection worldwide, particularly those that don’t have symptoms, have levels that are going to be below that. 

So you have an issue with invasiveness, the sensitivity issue and then there’s a few other specific issues. Currently, in sub-Saharan Africa, 80% of the rapid diagnostic tests that are used really target one antigen, the Hrp2 antigen, which is specific for falciparum. Unfortunately, there are now parasites, as we all know, that are circulating that have deletions and therefore escaping that diagnostic. 

These are key limitations that I think impact our ability to control and eliminate the disease.

So, these tests have their limitations – invasiveness, limited sensitivity, and the problem of deletions in key antigens because the parasites are evolving.

These limitations prompted researchers to rethink malaria diagnostics, leading to the development of a groundbreaking new test. It doesn’t detect parasite antigens from a sample of blood – rather, looks inside circulating blood to detect a byproduct of the parasite that all malaria parasites produce: 

Sunil Parikh: It’s something that all parasites produce. As opposed to some of the other diagnostic targets, it is something that the parasite is going to be unable to eliminate from its life cycle. So it’s a universal byproduct. After parasite invasion into a red blood cell, due to breakdown of hemoglobin by the parasite, you release heme, which is essentially an iron-containing substance. If that accumulates in large amounts, it becomes toxic to a cell and to the developing parasite. So over time, the parasite has developed an ability to package that iron into a crystal, an insoluble crystal, and that’s what hemozoin is, and that’s what you can see even under a microscope. It used to be called malaria pigment.

Hemozoin circulates in the bloodstream, inside red blood cells. The new test, the cytophone, uses lasers and ultrasound to detect it.

Here’s Professor Vladimir Zharov, a Professor at the University of Arkansas for Medical Sciences:

Vladimir Zharov: The cytophone is a relatively universal diagnostic platform which combines in vivo flow cytometry, laser technology and photoacoustic effect as a transformation of light energy into sound. 

The laser interacts preferentially with hemozoin inside infected red blood cells, whose absorption is higher than surrounding hemoglobins, which generate acoustic waves with specific signatures, parameters, such as width, polarity, shape and time delay that allows to identify signal origin, for example from malaria hemozoin compared to blood background hemoglobin.

When hemozoin absorbs a certain amount of the laser energy – emitted by the cytophone – it heats up and expands, generating ultrasound waves. The cytophone listens out for those ultrasound waves, indicating positive infection.

The technology was originally developed for the early detection and screening of cancer by looking for melanin, a marker for melanoma.

Like melanin, hemozoin can absorb a high amount of the laser energy, so is ideal for the test – also known as PAFC (photo-acoustic flow cytometry).

How does it work in endemic settings? Professor Yap Boum, Executive Director of the Institut Pasteur of Bangui in the Central Africa Republic, led a study testing the cytophone in Cameroon:

Yap Boum: In our environment in Cameroon, we still have quite a significant number of malaria cases, which happened mainly in children and mostly in the remote places where diagnostic and treatment might not be available.

The diagnostic of malaria, though a lot of improvement has been happening for this last decade, still it’s challenging to have accurate diagnostic of malaria, especially in the most remote part of our continent. So the PAFC [photoacoustic flow cytometry] came like a great opportunity for a non-invasive, which means we don’t have to prick. We don’t have to collect samples, which is an issue, but we can rely on a tool that can provide us robust results without collecting any blood sample. So this for me, as an idea, was already great.

In the test in Cameroon, the cytophone performed as well as current point-of-care diagnostic methods in individuals with confirmed malaria. Yap says some people were initially alarmed by the machine with all its connections and lasers – fearful it might burn – but gained confidence over time:

Yap Boum: The next step is to now assess people who have symptoms. Those people would go through a first assessment using the reference test, which is microscopy. Two, using rapid diagnostic tests that are validated by the World Health Organization so that they can be put on treatment for those having malaria. Three, having the PAFC test being collected, and four, to go even further to have some sample collected for the PCR. The PCR is the molecular test that is being done in Yaoundé, Cameroon, but also at Yale, so that we have two way of having reference so that we are confident enough on the result that will get out.

Those are the next steps – to test the sensitivity and portability of the technology;

Sunil Parikh: You know, this was the first first time we’ve tested it. This was a portable prototype, first time tested for this application in adults with confirmed malaria. We’ve just completed a study looking at healthy adults in Cameroon with a newer prototype version made by the Zharov lab to see how low this can detect because we know the majority of the people walking around with malaria are asymptomatically affected. So we should be able to really get a sense of the sensitivity, which I think will be much better than we demonstrated in this first study. This was really a proof of concept study. 

I think initially with a larger, you know, still portable version, you could imagine this being in kind of secondary tertiary level centers where you could use it for a lot of people, and you don’t really need consumables because the laser life is quite long. So in that case, you could use it as a real diagnostic. I’m sure a more smaller version that could be used in even more rural settings is possible, but I think, at least in my estimation, first off, we’d like to see this in more higher volume centers where this could be rapidly used. 

Vladimir Zharov: Right now we’re trying to improve our technology in two ways. First, to develop a portable device with advanced laser, transducer, software identification, and signal processing. Simultaneously we are developing a wearable prototype which is much cheaper, smaller, which theoretically provides continuous monitoring of infection progression. And if we talk about broad application, we continue the clinical trials with melanoma with a focus on early detection and screening, and in vivo detection of circulating blood clots in stroke patients with an ultimate goal to estimate the risk of stroke – either secondary or primary. But based on the combination of different technologies, we try to achieve our ambitious, challenging goal to detect a few bad cells, such as melanoma, malaria, or sickle or clots in the whole blood pool of approximately a few cells in five liters. This is what we are working on.

As Professor Zharov concludes, this interdisciplinary work is helping to make science fiction a reality:

Sunil Parikh: I think it’s an exciting technology. When I first talked with Professor Zharov, it almost seemed like science fiction to me. It wasn’t fiction to him because he’s a physicist and a bioengineer. it shows the importance of multidisciplinary collaboration and coming together from diverse perspectives to tackle and create innovative solutions.

We’ve been working together now since 2017 and was not easy to get to this point. A lot of people worked really hard. We pulled off this study in the middle of the Covid pandemic as well, which I don’t think we got a chance to talk about, but that was an additional challenge. But we learned as from our collaborators, who are amazing overseas in Cameroon, were able to really execute this study with minimal training.

The study in Cameroon indicates that the cytophone works. Using laser and ultrasound, rather than drawing blood, you can test for malaria by ‘listening’ for hemozoin in human veins. In the future, this might mean bloodless diagnostics…a leap forward in malaria diagnosis, and a testament to collaboration, innovation, and perseverance in the fight against one of the world’s deadliest diseases.

My thanks to Sunil Parikh, Vladimir Zharov and Yap Boum for being today’s guests.

You’ve been listening to the Johns Hopkins Malaria Minute. I’m Thomas Locke.