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Ohio State University Team Combines Immunoassay, Mini Mass Spec for Malaria Detection


NEW YORK – A team led by researchers at Ohio State University has developed a protein detection workflow that combines paper-based immunoassays with miniaturized mass spectrometry to enable diagnostic testing in resource-constrained areas.

In a study published this month in Analytical Chemistry, the researchers used the technology for malaria detection and suggested it could be applied for large-scale surveillance and screening for the disease.

The method makes use of ionic probe technology developed by Abraham Badu-Tawiah, professor of chemistry at OSU and the senior author on the study. According to him, these probes provide a more stable alternative to reagents used in conventional immunoassays, making them potentially useful for work in areas where cold storage is not widely available.

Initially described in a 2016 paper published by Badu-Tawiah and collaborators in the Journal of the American Chemical Society, the probes remain stable for several weeks under ambient conditions and are designed to release, upon treatment with ammonium hydroxide, mass tags that can be detected using paper spray ionization mass spectrometry.

In their malaria work, Badu-Tawiah and his colleagues used the technology to detect the malaria antigen Plasmodium falciparum histidine-rich protein 2 (PfHRP2), building a paper-based immunoassay that features a detection antibody linked to the ionic probe. They collected blood samples and ran the immunoassay, and when they were ready to read out the results, they treated the paper to release the ionic probes' mass tags, which were measured using a portable mass spectrometer. They found that they could store the paper immunoassay after running it for up to 25 days at room temperature without affecting the clinical results.

The approach could enable low-cost, high-throughput, and highly portable malaria testing in low-resource regions, Badu-Tawiah said.

"I wanted to develop a universal approach" to malaria testing, he said, noting that he aimed to develop a system that could be used at the point of care for diagnosis of symptomatic individuals as well as for population screening and management of outbreaks.

Currently, several different technologies are available for diagnosing malaria, but each has drawbacks, Badu-Tawiah said.

PCR, for instance, is highly sensitive and specific, but it requires upfront sample processing and specialized equipment. While point-of-care and home PCR testing has come to market in recent years, these assays are quite expensive.

There are also rapid antigen tests for malaria, but these tests can be expensive, too, Badu-Tawiah said. He noted, for instance, that Abbott's BinaxNow Malaria tests can cost more than $40 each, though his co-author Kingsley Badu, leader of the Vector-Borne Infectious Disease Research Group at Ghana's Kwame Nkrumah University of Science and Technology, said that less expensive alternatives from companies like Biocredit can cost as little as $1 per test.

Microscopy is the traditional gold standard for diagnosing malaria, but, as the study authors wrote, it is "labor intensive" and "subjective" as well as "technically challenging."

By contrast, the paper-based immunoassay used by Badu-Tawiah and his colleagues is inexpensive and requires no sample processing. And because the readout relies on mass spec-based detection of the stable ionic probes — as opposed to enzymes or fluorophores, both of which can quickly degrade — samples can be collected, the immunoassay can be run, and the results analyzed weeks later. That means, for instance, that health workers doing surveillance in remote areas could collect samples from hundreds or thousands of people at one time and analyze them later — either by sending them to a central lab for mass spec analysis or by using a battery-powered portable mass spectrometer.

Badu-Tawiah recently returned from Ghana where he said he was able to collect malaria testing samples from 500 individuals by himself.

In the Analytical Chemistry study, the researchers used a wax-printed paper immunoassay that included a number of preparation steps, but they have since developed a more streamlined device that Badu-Tawiah said only requires users to deposit a drop of blood and then add buffer. The immunoassay portion of the test takes around 15 minutes, while the mass spec readout of the ionic probe mass tags takes around a minute per sample.

While the immunoassay costs around $.50 per test, the mass spec instrumentation is considerably more expensive. In the study, the researchers used a Continuity miniature mass spectrometer from San Jose, California-based BaySpec, which Badu-Tawiah said costs around $150,000. He noted, though, that this is a one-time cost and that many thousands of people could be screened using the same instrument.

Kwame Nkrumah University's Badu said the method could prove particularly useful as countries move from a focus on controlling malaria to efforts to eradicate it. Such a shift, he said, will require extensive surveillance testing to identify asymptomatic carriers who should be treated for the disease.

In fact, Ghana is in the midst of such a shift, he said, noting that several days ago, the National Malaria Control Program decided to officially move from the control to the elimination phase.

"So we believe that it is a very opportune time for new tools like this to be able to help," Badu said.

He said that he and Badu-Tawiah have engaged with the Ghanian government and are working to demonstrate that their approach offers improvements in performance and cost over existing methods — primarily rapid antigen testing. Badu-Tawiah is currently evaluating the 500 samples he collected in Ghana, and the researchers are in the process of collecting additional specimens, with the aim of reaching 800 — half from symptomatic and half from asymptomatic individuals.

In the Analytical Chemistry study, the test was able to detect the malaria antigen PfHRP2 in untreated human serum at levels down to 0.216 nmol/L, below the 0.227 nmol/L sensitivity threshold recommended by the World Health Organization for evaluating symptomatic patients, though the study presented data from only six samples. In terms of cost, Badu said the test is currently more expensive than the low end of the rapid antigen market but he expects costs will come down once the test is fully developed and being manufactured at scale.

Jonathan Cooper, a professor of engineering at the University of Glasgow whose research focuses in part on the development of diagnostics — including for malaria — in resource-constrained settings, raised a number of concerns about the approach, however.

In an email, he noted that the clinical validation looked at only a small number of samples, running them on a mass spec instrument in a centralized facility with sample preparation done in advance, and achieved only "relatively low sensitivities" that, while sufficient to detect malaria in symptomatic individuals, would not be enough to detect the disease in asymptomatic people — as would be required for a disease elimination strategy.

He said that other challenges to the method include whether complex instruments like mass spectrometers "can be used and maintained in rural underserved communities, close to the point of care/point of need."

Additionally, he noted that deletions in the Plasmodium hrp2/3 gene, which expresses the malaria antigen PfHRP2 measured in the assay, are becoming more common. These deletions would allow the pathogen to escape detection by the current test.

"It will be interesting as to whether the authors can adapt their existing platform to take into account this current evolutionary pathway," Cooper said.

"The approach of combining paper microfluidics for antibody-based sample enrichment with mass spectrometry is interesting and serves to highlight ongoing challenges that remain in malaria diagnostics, and how highly sensitive, low cost, and easy-to-use tests can be used in community settings," he said.

Regarding the practicality of using mass spectrometry at the point of care, Badu-Tawiah said that BaySpec, which makes the instrument used in the Analytical Chemistry study, has a training program in which it teaches nurses to use the system.

"It is not difficult at all to use this instrument," he said. "Certainly, Ghana has the manpower to run it."

That said, he noted that a "central analysis model" where samples are collected on the paper-based immunoassays and then sent to a central lab "is our best option" as it would allow for a more efficient sharing of resources.

He suggested that, for instance, mobile money transfer facilities, which are widespread throughout Ghana, could make for good locations for dropping off and picking up tests.

Badu-Tawiah suggested that portable mass specs could prove useful even within a centralized model because of how robust and easy to maintain they are compared to larger instruments.

"Bigger mass spectrometers need to run continuously to maintain performance. Not so with portable instruments," he said. "The portable mass spectrometer is robust — turn it off when not in use, and turn it on when needed. It takes only 10 minutes to get it ready for analysis."

Badu-Tawiah acknowledged the issue Plasmodium hrp2/3 gene deletions present for the test but said this could be addressed by including additional markers like Plasmodium lactate dehydrogenase (pLDH), which he said the WHO has begun recommending being included in malaria tests. He also said that the paper-based immunoassay-mass spec test is amenable to multiplexing.

Badu-Tawiah also acknowledged the study's small sample size and said that he is now preparing to evaluate the assay using the 500 samples he recently collected in Ghana. He added that in unpublished work, he and his colleagues have significantly improved the sensitivity of the test to where it could potentially be used for diagnosing asymptomatic individuals.

In addition to malaria, the researchers are developing the technology for measuring markers of other infectious diseases including leishmaniasis. Badu-Tawiah said he is also exploring use of the ionic probes for measuring cancer markers.

He said he has ambitions to commercialize the technology, but has yet to do so, in part due to the difficulty of getting investors excited about an application like malaria testing and surveillance, which is unlikely to yield large profits.

"It's difficult to get investors who are interested in malaria," he said. "I mean, every investor wants some money back, right?"