NEW YORK (360Dx) – A group of Texas State University (TSU) researchers have developed a rapid, microfluidic-based immunoassay to rapidly detect gastrointestinal infections (GI) in patients.
Partnering with Austin, Texas-based Paratus Dx and its smartphone-based PreparedNow spectrometry platform to further develop and commercialize the assay, the group hopes to diagnose cases in developing nations and areas lacking hospitals and point-of-care (POC) centers.
Shannon Weigum, a biology associate professor at TSU, explained that her team originally wanted to build a custom panel to capture and identify pathogens linked to GI.
"GI and diarrheal diseases are a very large global health problem, as the number of children in developing areas that die every year is staggering," Weigum said. "Yet, there's little done to diagnose the disease, which leads to reinfection and spread of pathogen that continue over and over in a cycle."
Weigum highlighted that her lab focuses on infectious disease assay development by identifying and characterizing antibodies to see which are the most appropriate for certain settings based on their kinetic properties. The team aims to detect pathogens that cause GI and other ailments that cause similar symptoms, such as rotavirus, norovirus, and protozoan parasites common in remote regions.
Weigum said that her team is now developing an assay that applies a "sandwich-based" lateral flow method to identify targeted pathogens. Researchers first use a swab to collect about 200 to 250 μl of a patient's fecal sample. Inserting the swab into the microfluidic kit within the credit card-sized PreparedNow system, the team uses delivery secondary reagents and a tagged antibody that specifically binds to the target. The microfluidic device uses capillary-driven flow materials and embedded-specific capture reagents within nitrocellulose with the antibodies forming a "sandwich" in the presence of the pathogen.
According to Weigum, the system then uses a chemiluminescent tag that reacts to form a chemical reaction and generate light in the presence of the target pathogen. The PreparedNow platform then applies the smartphone's camera as a spectrometer that collects light and analyzes it for different wavelength properties and intensity information.
As part of the system, Weigum and her team embedded multiple antibodies that each targets a specific pathogen in an array pattern to increase detection of diverse pathogens within a single sample. Weigum explained that they needed to apply antibodies that had dissociation constants — which act a measure of antibody and affinity and "tight" binding — in the nanomolar range. By developing a multiplex assay, she believes her team can narrow down detection by collecting all pathogens in a single assay strip.
While the researchers initially developed the GI assay on a paper-based microfluidic platform at Weigum's lab at TSU, the team has since switched the assays over to Paratus Dx's PreparedNow POC system. Weigum said the immunoassay can produce diagnostic results in about 20 minutes, with the smartphone displaying a bar graph that shows the quantitative amount of the bacterial, viral, and protozoan parasites. However, she declined to comment on the assay's clinical sensitivity or specificity.
"What's interesting is that because the [PreparedNow] is a universal platform, assays can be developed for different syndromes," Weigum said. "All that is needed is to change out the assay strips and internal immunoassay reagents for each panel."
According to Weigum, her team began collaborating with Paratus Dx after she met CEO John Carrano at the 360 Global Health Technologies Design Competition at Rice University in 2014. She said that they both saw the potential in improving the gap in automated sample preparation for infectious disease diagnostics and decided to work together. After applying and receiving a Phase I Small Business Innovation Research Grant in 2017, Paratus opened a wet lab at Texas State University's Science, Technology, and Advanced Research Park.
Weigum noted that one of the major challenges of general POC detection is the lengthy sample prep process, which can require the user to perform several intricate and complicated steps. By porting the assay onto Paratus Dx's PreparedNow system, she believes that the team can simplify the workflow and expedite the diagnostic process. Since working with Paratus Dx, Weigum's team has begun developing an additional set of assays to detect specific GI bacterial, viral, and protozoan pathogens.
However, Weigum highlighted that the cost of testing specific antibodies has been expensive. Her team has tested several reagents for the assay by examining their full characterization and kinetic profile. The researchers initially screened several antibodies and then narrowed in on the ones they believed would perform the best.
"The protocols can be very expensive since each single antibody can cost up to a few hundred dollars to select them," Weigum said. "When you want to screen 20 different antibodies, that can get pretty costly."
Weigum said that while her team debated applying for an IP for the technology, they decided not to follow the route because other academic groups could "potentially adapt the system by swapping out a reagent or piece of the system and apply for a similar patent." Her team, therefore, chose to keep the assay a trade secret. On the other hand, Paratus Dx has received six patents related to the PreparedNow system, in addition to 12 more pending patents.
While Weigum's team works to drive down the cost of the assay and improve its clinical sensitivity and specificity, Weigum believes that the assay could be eventually used in developing countries that deal with chronic cases of GI disease. The team's overall goal is to detect six to eight pathogens that are linked to the disease.
In addition to the SBIR Phase I grant, the group has received funding from the US Department of Defense through a two-year $2 million SBIR Phase II grant in 2018. Carrano said that the funding will support research and development, as well as commercialization.
Noting that the microfluidic chip is past the proof-of-principle stage, Weigum said that her team wants to expand the number of pathogens they are testing as part of the system.
"When you're multiplexing, that puts additional constraints on reagent selection to ensure minimal cross reactivity between them," Weigum said. "The next challenge is to ensure that we can continue to [accurately] multiplex up to a certain number of pathogens with those reagents."
According to Carrano, the PreparedNow platform is still under development, with an expected full commercial launch in 2020. Following the ParatusDx's launch, the group aims to seek 510(k) approval from the US Food and Drug Administration and hopefully launch the GI immunoassay in 2021.
Weigum said that her team aims to begin preliminary preclinical steps by working with Dell Children's Medical Center in Austin later this year. Weigum's group will perform preclinical test runs to ensure appropriate sensitivity and specificity, and eventually compare the assay's performance with Biofire's FilmArray GI Panel.
In addition to Biofire and its FilmArray technology, other groups and firms developing or offering similar platforms to rapidly diagnose GI pathogens include Becton Dickinson, which offers a rapid molecular diagnostic assay that detects infectious diarrhea caused by intestinal bacteria as an extension of its BD Max qPCR panel.
Molecular diagnostics manufacturer GenePOC is now developing a specific "SMRT" GI assay for its Revogene multiplex system, which uses centrifugal microfluidics and consumable cartridges to detect up to 12 targets.
Sandia National Laboratories researchers have also developed a syndromic assay that integrates immunoassays and isothermal amplification molecular testing to identify GI pathogens. Running on the firm's SpinDx system, the assay can detect three protein-based enterotoxins and DNA from three enteric pathogens in less than an hour.
In contrast to GI platforms like Biofire's panel, Weigum said that running the assay on Paratus is low-cost and does not require an expensive, dedicated instrument besides a smartphone. When the group launches a commercial version of the assay, Weigum said that that each assay will cost the end user less than $10.