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Sandia Researchers Combine Molecular, Immunoassays for Syndromic Testing


NEW YORK (GenomeWeb) – With the aim of diagnosing gastrointestinal infections causing diarrheal disease, researchers at Sandia National Laboratories have developed a syndromic test that combines immunoassays and isothermal amplification-based molecular testing. Essentially a small, fully integrated syndromic panel, the test can go from stool sample to result using the next generation of a platform developed at Sandia called SpinDx.

Described in a paper published in August in Biosensors and Bioelectronics, the assay can detect three protein-based enterotoxins as well as DNA of three enteric pathogens. Specifically, it detects cholera toxin, Staphylococcal enterotoxin B, and Shiga-like toxin 1, as well as Campylobacter jejuni, Escherichia coli, and Salmonella typhimurium, in less than one hour.

There are approximately 1.7 billion cases of diarrheal disease globally each year, and the syndrome is frequently caused by infections. Children are particularly vulnerable; diarrheal disease kills more than half a million children worldwide each year, and is the second leading cause of death in children under the age of five according to the World Health Organization. It also represents a significant unmet diagnostic need, particularly in the developing world.

The Sandia test runs on the latest iteration of the SpinDx system, a centrifugal microfluidics diagnostics platform with integrated sample preparation. Among other enhancements, the newest version now incorporates non-contact temperature control using a medium-wave infrared heater.

"That allows us to do molecular diagnostics," Chung-Yan Koh, a senior research scientist at Sandia and a corresponding author on the study, said in an interview.

SpinDx uses centrifugal force to propel sample through microfluidic chambers containing reagents. It was developed originally for multiplex immunoassay applications, but in 2016 Koh indicated in an interview that his team was working on a project to create a combined system.

The molecular testing method the Sandia group ultimately decided upon was loop-mediated isothermal amplification, or LAMP. They ruled out qPCR because the thermocycling is too power-intensive for their long-term goal of making the system deployable. They also explored other isothermal methods such as recombinase polymerase amplification, or RPA, but given that another goal was to make the system as close to sample-prep free as possible, they ruled out RPA because "it requires a minimal amount of nucleic acid purification," such as binding to silica and a shock heat flash step.

"We wanted to make it even more user friendly," Koh said.

The LAMP technology is also very resistant to traditional PCR inhibitors found in some clinical sample types. To further enhance this resistance, the Sandia team incorporates a proprietary technology called quenching of unincorporated amplification signal reporters, or QUASR, which uses quenchers instead of intercalating dyes to "get better sensitivity and selectivity, with a decrease in false positives," Koh said.

On the immunoassay side, the team has also added integrated sample distribution and metering, he said.

The upshot of these tweaks is that the instrument can essentially perform sample-in, answer-out testing with stool, a notoriously difficult sample type for molecular testing. The test requires one up-front step to dilute the sample before it can be pipetted into the system, but Koh said the group is currently working to eliminate that step.

The Sandia system is also over-engineered in terms of preventing contamination, Koh said. Disks are sealed in several ways to prevent release of vapors and particles, although they are not air tight because the samples are heated and need to be able to vent dissolved gasses. There is also a workflow step if a user is concerned about potential contamination which can put in a delay, preventing the interlock from releasing until all aerosols are settled.

The instrument is being developed on the research scale, so it is not so easy to determine what the cost would be to produce it, Koh said, but it uses many common off-the-shelf components that are listed in the study.

Diagnostics systems using spinning disk technologies are not new but seem to be gaining some traction of late. For molecular testing, commercially available technologies with US Food and Drug Administration-cleared assays include the Revogene instrument from GenePOC and Liaison MDX from DiaSorin.

A spinning disk-based system called MicrobeDx developed in the lab of Marc Madou at the University of California, Irvine recently won $3.5 million in funding from CARB-X. And a system called LabDisk was spun out of the research institute Hahn-Schickard to form SpinDiag, which raised €1.6 million ($1.9 million) in seed funding last year.

For non-molecular testing, the Piccolo Xpress from Abaxis can perform a variety of chemistry panels — some of which are CLIA waived — from a few drops of blood using a disk-based system, and the GyroLab system from Gyros Protein Technologies can be used for immunoassay testing as well as test development.

A handful of others have combined molecular tests and immunoassays into a single platform, including the DiagCore instrument from Qiagen's StatDx, a platform from Rheonix, the Luminex xMap system, and a system being developed by researchers at the University of Pennsylvania, for example.

However, the Sandia system appears to be the first to incorporate spinning disk-based technology with a combined immunoassay and molecular test.

Overall, Koh said the system could someday prove very useful for near-patient syndromic diagnostics. Querying the protein space can be useful for toxins, or for deducing history of exposure through antibody and antigen tests, he said, adding that for national security and public health applications, the protein itself may also be more important than the causative organism. Molecular diagnostics, on the other hand, provide better sensitivity as well as species-level identification.

"By combining the two, you could potentially end up with a much more confident diagnosis at the end of the day," Koh said.

The next steps for the Sandia team involve making the system "truly portable" by making it more rugged, hardened, and miniature, with a lighter-weight battery, so that it could go into a defense or first-responder situation. Koh and his team are also trying to come up with better ways of doing the sample metering across the disk, reducing it from dual-inlet to a single inlet for sample input. They are also considering potentially converting the reagents on the consumable to a dried format and performing shelf-life studies for different operational environments.

The SpinDx system has been exclusively licensed for specific fields of use, such as by drug testing manufacturer LifeLoc, and it is also being used by co-inventors of SpinDx in a spinout firm, Sandstone, for an FDA-cleared at-home male fertility testing device called Trak. The technology is also still available for licensing.