Malaria’s spit solution

Dr. Dinglasan recently received a $1.3 million grant to bring a prototype rapid-diagnostic test for malaria to commercial viability. The test uses saliva, which eliminates the dependence upon blood of all other current tests; and it is able to detect “subclinical” infections — in other words, it can diagnose malaria even before someone feels really sick. Getting subclinical infections treated is key to eliminating malaria because these infections form a reservoir that fuels transmission back into mosquitoes when they take a blood meal

A new $1.29 million grant from the Global Health Innovative Technology Fund will support EPI malaria investigator Rhoel Dinglasan’s work to develop a novel saliva-based malaria diagnostic test that catches infections even when victims don’t show symptoms.

When Rhoel Dinglasan, Ph.D., hears malaria researchers from the generation before him say that it’s impossible to eliminate the age-old mosquito-borne disease, he responds: “Let my generation try. Don’t close the door before we are even given a chance.” 

Dinglasan, an associate professor of infectious diseases with UF’s Emerging Pathogens Institute, began his career by researching a new type of malaria vaccine and investigating target compounds for drug discovery. But after witnessing the struggles of malaria-affected families in Cameroon while conducting field work between 2011 and 2014, his path veered into improving disease detection.

Portrait of Rhoel Dinglasan.
Rhoel Dinglasan, Ph.D, M.P.H. and M. Phil., shown in his EPI laboratory. His home academic department is in UF’s department of infectious diseases and immunology in the College of Veterinary Medicine.

“I want to make a malaria test that Dr. Mom or the local school teacher can use,” Dinglasan says. “Without drawing blood, pricking fingers, needing microscopes, or trekking to the community health clinic that’s ten or fifteen miles away and might lack water.”

While working in regional African clinics, he observed the emotional trauma that drawing blood elicited in kids, and — after his then-five-year-old daughter melted down and began drooling during a pediatric appointment at the prospect of shots — he felt compelled to find a needle-free, bloodless way for people to test themselves for malaria.

After all, if a woman can learn whether or not she is pregnant in the privacy of her own bathroom, why shouldn’t people be able to painlessly screen themselves for malaria whenever and wherever needed? But it was a stray nick during Dinglasan’s next dental hygiene appointment that gifted a gem of an idea: What if the answer to a new malaria diagnostic test lay hidden in the victim’s saliva?

Tests at their limits

Malaria kills an estimated 435,000 people every year, most in sub-Saharan Africa, and there are an estimated 217 million cases annually, according to a 2018 World Health Organization report. Those most at risk are children under the age of five. Twelve years ago, the Bill and Melinda Gates Foundation announced that one of its goals was global malaria eradication. Detection and treatment are two key strategies to halt the cycle of Plasmodium parasites moving between human and mosquito hosts.

Warmed culture medium awaits the addition of malaria parasites for study in the Dinglasan lab.

While microscopy of blood samples is the gold-standard diagnostic, it is not available in a many rural areas where malaria is endemic but technology is not. Rapid diagnostic tests offer an inexpensive way to quickly test many people. But current RDTs are running into both biological and pragmatic limits. Most tests work by screening someone’s blood for a biomarker protein, histidine-rich protein 2, which is secreted by Plasmodium.

But multiple paths can lead to a false negative, which has undercut the test’s reliability and allowed the disease to spread even where intense control efforts are underway. “The WHO recommends confirmatory diagnosis by rapid diagnostic test that someone has malaria before triggering the drug,” Dinglasan says. “But those who don’t come up positive, we miss. That parasite then doesn’t get treated and killed. And it spreads and spreads.”

Most striking, histidine-rich protein 2 is dropping out of some parasite populations due to a genetic mutation. “Malaria researchers began to notice in 2012 and 2013 that some parasites in Africa no longer carried this gene for the secreted protein biomarker,” recalls Dinglasan, who also directs the CDC’s Southeastern Regional Center of Excellence in Vector Borne Diseases. “And everyone was like ‘No, no, no; that’s only happening in Eritrea.’ But now these mutated parasites that lack this gene are popping up all over the continent. It spread. What used to be the minority population of parasites could potentially be the majority population in the future.”

Vincent Nyasembe, Ph.D., a postdoctoral fellow from Kenya in the Dinglasan laboratory, extracts RNA from mosquito samples for downstream projects where gene expression will be reduced, also known as gene knockdown studies

Second, even without the hassle of this genetic mutation, current tests miss cases when the parasites are in a human host but are either not well represented in the blood (a low-density or sub-clinical infection), there is an excess of parasites (a high-density infection), or when the victim’s immune response produces an overabundance of antibodies (the prozone effect). In sub-clinical cases, people may have developed some level of partial immunity which allowed their body to fight off some of the parasite, but they still carry a low density. They may not experience classic symptoms or even feel very unwell.

“This is a huge problem, because people who are legitimately infected may be misdiagnosed based on a false negative, and given antibiotics or some other type of drug that won’t help them with malaria,” Dinglasan says. “Or, if they don’t feel sick, they won’t even seek treatment. These untreated cases continue the cycle of transmission.”

Because they carry parasites, these victims act as a vast and unwitting reservoir: it is they who are now spreading the disease to mosquitos.

Rethinking the problem

Rather than viewing these limitations as a stop sign, Dinglasan sees opportunity: the chance to rethink how we test for malaria. New biomarkers needed to be identified, and breaking down the mental and cultural barriers to sampling blood wouldn’t hurt either.

“Working with blood doesn’t help us to break out of clinical settings,” Dinglasan says. “So, saliva was it.”

After his ah-ha! moment in the dental hygiene chair, he researched whether it was feasible to detect Plasmodium parasites via biochemical traces in a human host’s saliva. A prior study had detected Plasmodium DNA in saliva and urine, but it used only suspected clinical cases of malaria, which didn’t help in addressing the subclinical reservoir of infected people who fuel transmission. Dinglasan wanted to find a protein biomarker, which is very different than testing for DNA, and it had to also work in asymptomatic carriers to have the biggest public health benefit.

A smart phone camera captures lateral flow prototype test results through a custom-tailored imaging box with a filter. The fluorescence signal results from excitation of the europium chelate detection particle which is bound to a biomarker on the test (T) line. The detection particle is activated by an ultraviolet -flashlight, shown inserted into one end of the 3D-printed imaging box. The control (C) line shows that the test is working. Pictured: Tim Hamerly, Ph.D., of the Dinglasan laboratory.

He studied dental textbooks, and human physiology, and also revisited what is known about Plasmodium biology. “I had a hunch that molecules secreted by the parasites might follow the same conduits as our immunoglobulins into our saliva,” Dinglasan says. Specifically, he followed previous findings from the 1980’s – and a study in rodents by one his mentors, professor Bob Sinden at Imperial College London — and further theorized that when the parasites are in the mature gametocyte stage they gather in capillary beds in gum tissue. Gametocytes do not produce clinical symptoms, but they ensure that the parasite jumps from humans back into mosquitoes.

Bit by bit, Dinglasan stitched together evidence into a testable hypothesis: Plasmodium gametocytes secrete proteins, tied to an evolutionarily-conserved gene, that should be detectable in human saliva before a person even feels ill. The Gates Foundation stepped in with $110,000 to allow Dinglasan to find concrete answers and he soon confirmed his hunch. It was a finding that broke his focus away from malaria vaccine research and set him on a new path.

At the time, Dinglasan was working at John Hopkins and he soon entered the Maryland Innovation Initiative to compete for a $100,000 grant which would allow he and his team to patent his idea. The program linked him with President Obama’s D.C. Innovation Corps, which he credits with turning on its head the whole idea of making new biotech. “They actually asked, ‘So you really think you know that your technology is wanted by people in the field? Have you considered that maybe it isn’t?’” Dinglasan recalls. “They challenged us to interview twenty-five stakeholders all around the globe, within a week.”

The challenge proved pivotal. His team modified their prototype test based on the wish-list of features asked for by clinicians and end-users. “We just didn’t know what they wanted until we asked,” he says. Improvements included: a positive control to test bulk orders that may sit unused for lengthy time periods, making the test quantitative, and providing software that works across multiple cell phone operating systems. This is needed to better manage test result images for patient records, diagnostic validation and malaria control program reporting standards.

Hamerly is maintaining Plasmodium falciparum (malaria parasite) in cultured human red blood cells. The work is isolated in a biological safety cabinet in Dinglasan’s BSL2 laboratory. These parasites are used as baseline biological material for all of the lab’s studies, ranging from genetics to metabolomics to experimental infections of mosquitoes.

Given nine months to come up with a prototype, his team delivered. “Then we were like, ‘Now what?’ ’” Dinglasan recalls. “What are the next steps to make this commercially available?”

But because the technology they were developing competed with interests of other well-established malaria researchers, the team ran into lengthy delays getting their work published. “It was a long, hard fight, but we won out,” Dinglasan says. Their work was published in Science Translational Medicine in January 2019. Eight years had passed since his field work in Cameroon first got him ruminating over how to better test for malaria in endemic areas. 

Although the critics argue malaria diagnostics should be used primarily in clinical settings, Dinglasan countered to remember the parasite’s biology: it is only abundant and circulating in the blood at certain stages of its lifecycle. At other times, it hides in the liver or bone marrow and sequesters in the vasculature and certain types of capillary beds such as those located in the gingiva. A superior test would not be limited to detecting a parasite or its proteins in blood, but could also detect parasite proteins no matter where the parasite was in the body. 

“And guess what?” Dinglasan says. “Ours does that.”

Applying research to the real world

Dinglasan’s team is focused on moving their research into the hands of end users who will benefit. He is working with several biotechnology businesses to translate the saliva-based detection technology into a viable commercial form. The prototype is the size of a shoe box, and needs to be engineered down to the size of a pregnancy test, scalable and available at a $1 price point, or less.

The instrument at the heart of malaria parasite biomarker discovery in Dinglasan’s lab: a mass spectrometry unit used to detect and identify proteins and metabolites.

To fund this transition from lab bench to real world, the Japanese Global Health Innovative Technology Fund recently awarded he and his partners $1.29 million. The GHIT Fund selects research and development projects for support based on the likelihood that the technology, pharmaceutical or vaccine will help lift people globally from the burden of infectious diseases associated with poverty.

The Saliva-based Malaria Asymptomatic and Asexual Rapid Test or SMAART-1, will be developed in conjunction with two Japanese biotech companies: CellFree Sciences, and Frontier Institute, plus U.S.-based Oasis Diagnostics, and South Africa-based ERADA Technology Alliance.

SMAART-1 improves upon current RDTs by detecting asymptomatic carriage of Plasmodium with increased sensitivity.  Similar to current technology used for rapid detection of malaria in clinical settings, the new SMAART-1 test is based on a lateral flow design. This means that as the liquid sample is pushed across a cellulose test strip, the device screens for the direct presence of Plasmodium female sexual stage protein 17 (PSSP17) biomarker. This protein is found across both P. falciparum and P. vivax species and current data suggest that it is not likely to drop out of any one population because it is an evolutionarily-conserved and essential gene.

The saliva-based test’s sensitivity to PSSP17 approaches the precision of polymerase chain reaction tests. Whereas some current blood-based RDT’s can only detect 100 asexual parasites in a microliter of blood, the saliva-based test is sensitive at one to 10 female gametocytes per microliter of blood. And if someone is carrying sexual stage gametocytes, then they are also playing host to asexual stage parasites.

Dinglasan laboratory members pictured at the EPI building: (Left to Right) Kaci McCoy, Borja Lopez-Gutierrez, Rhoel Dinglasan, M.Phil, Sazzad Mahmood, Tim Hamerly, Nicole Bender, Vincent Nyasembe and Prachi Khar. Not pictured: Heather Coatsworth, Anna Dell, Rosie Steck, Alexandra Leyte-Vidal, Caroline Stephenson and Jasmine Ayers.

The research team will design the test for use in children as young as two up to adults with readable results ready in 20 minutes or less and a shelf life of 30 months. Oasis Diagnostics will produce at least 1,000 tests for pre-clinical testing and the GHIT grant will also support development and field-testing of instructional pamphlets in English and French in either  Uganda or the Democratic Republic of the Congo. ERADA Technology Alliance holds the patent license and will shepherd the test through commercial development.

If Dinglasan’s team and his biotech business collaborators can re-engineer the prototype into a commercially available test, then it could be a game changer in how countries strategize for malaria elimination. Detecting and treating subclinical infections is a necessary part of reducing the transmission reservoir until no cases are left.

“In the first year of this grant, we will make the saliva-based test commercially viable,” Dinglasan says. “Shortly after, we will make a test with the same biomarkers to also work with blood.”

And on hard days, when it feels like it’s taking too long to reach these goals, he reminds himself of his lab’s mantra: “Nothing in life that is worth doing is easy.”

By: DeLene Beeland