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KGI, Claremont View Mechanical Sample-Prep Cartridge as Key to Range of POC Infectious Disease Tests


Scientists from the Keck Graduate Institute of Applied Life Sciences and KGI spinout Claremont Biosolutions have been developing point-of-care molecular testing platforms and associated assays for Clostridium difficile, multi-drug-resistant tuberculosis, and herpes simplex virus, representatives from both organizations said recently.

The groups are developing some of these platforms in collaboration and others separately, using different nucleic acid amplification and detection schemes. However, all the platforms will use a common sample-prep cartridge that integrates technology developed by both groups and that performs mechanical lysis of bacterial cells or viruses and non-chemical nucleic acid extraction at a low cost, which is expected to significantly drive down the overall cost of the final systems.

Angelika Niemz, director of research at KGI, provided an update on her group's work at Cambridge Healthtech Institute's Future Diagnostics conference, held last week at the University of California, Irvine.

Niemz framed her presentation around her lab's attempt to develop a truly self-contained, inexpensive, disposable, handheld, sample-to-answer nucleic acid testing device for diagnosing tuberculosis and MDR-TB in the developing world.

She pointed out that one of the most successful molecular diagnostic tests for TB, Cepheid's GeneXpert MTB/RIF, is a "great" test, but that the discounted rate of around $17,000 for the GeneXpert platform under Cepheid's high-burden developing country program is still too expensive for many settings, especially considering the cost of annual maintenance and the need for an air-conditioned laboratory to run the platform.

In contrast, Niemz's group – like many other research groups and diagnostic companies – has been exploring isothermal amplification technologies, as opposed to real-time PCR, to simplify the workflow and reduce the cost of a diagnostic platform by eliminating the need for a thermal cycler.

One technology that the KGI group has been exploring is loop-mediated isothermal amplification, or LAMP, which is owned by Eiken Chemical but used by multiple organizations in molecular diagnostic development, the most successful of which may be Meridian Bioscience's Illumigene-brand tests for C. difficile and group B Streptococcus.

Another isothermal technology — exponential amplification reaction, or EXPAR — was originally developed by KGI scientists, who first described it in a paper in the Proceedings of the National Academy of Sciences in 2003.

According to Niemz, EXPAR is a fast isothermal reaction that uses nicking enzymes to cut recognition sites adjacent to a particular target to achieve target-specific amplification in minutes at a constant temperature of around 60° C.

The KGI group is exploring the use of lateral flow strips — another relatively inexpensive technology — to detect amplicons from both isothermal amplification methods.

However, according to Niemz, the ultimate key to creating a truly inexpensive point-of-care device may be the integrated sample-prep cartridge featuring technologies originally conceived of at KGI and licensed in 2008 to Claremont Bio, which further refined them into commercial products. Claremont spun out of KGI in 2006 and is located in nearby Upland, Calif.

The group's prototype combines mechanical cell lysis and DNA extraction technologies into an injection-molded cartridge that is about the size of an iPhone and includes thermally bonded film pouches — a "retention pouch" and a "pump pouch," Niemz said.

Claremont Bio currently sells the cell lysis and DNA extraction technologies under the brand names OmniLyse and PureLyse, respectively. But, according to Robert Doebler, president and co-founder of the company, the integrated cartridge features proprietary valve technology and electrolytic pumping "so you can now, in an automated fashion, flow sample through the same chamber, turn the valve … and do the wash and the elution."

The technology is different from many other nucleic acid sample prep solutions because "essentially we've miniaturized mechanical cell lysis," Doebler told PCR Insider this week. "There is a micromotor embedded … inside a valve. Our [intellectual property] lies in the fact that we're lysing mechanically — so there's no chemical processing of cells, which eliminates all the wash steps associated with chemical lysis. Now you have a very clean lysis step that can be done in under a minute … and we are currently binding nucleic acid to the same media that is being used to help lyse the cells. So we're killing two birds with one stone."

Niemz noted during her presentation last week that the current cost of an integrated cartridge is around $15, but that the group would like to reduce that cost even further by developing next-generation valving technology.

POC Testing Platform

Niemz is currently principal investigator on a pair of grants awarded to KGI by the National Institute of Allergy and Infectious Diseases to support the development of a testing platform that would integrate the sample prep cartridge with various aforementioned isothermal amplification and lateral flow detection technologies for rapid diagnosis of infectious diseases.

The first grant, which runs through June 2014 and is worth $847,000 in its first year, supports the development of an integrated device for rapid diagnosis of drug-resistant TB from sputum at the point of care. The cost of the envisioned system, according to the grant's abstract, will be 10 to 100 times lower and the size "significantly smaller" than current fully integrated, PCR-based benchtop systems.

The second grant, which is worth $540,000 and will end this June, supports development of an integrated device to enable rapid diagnosis of herpes simplex viruses I and II in STD clinics and maternity wards.

"It turns out [the sample prep cartridge] does work with viruses," Claremont's Doebler said. "It doesn't take as much to disrupt the virus to get the nucleic acid out, but it doesn’t hurt it. In fact, it helps. You eliminate any kind of diffusion limitations because … the beads are moving, and binding is very efficient and fast."

Likewise, Claremont was previously awarded two separate Phase I Small Business Innovation Research grants from NIAID, worth about $300,000 each, to develop its own integrated molecular testing device for detecting the TcdA gene, TcdB gene, and TcdC point mutations of C. difficile at the point of care from stool samples and environmental swabs.

Doebler said that although those grants have expired, the company is hoping in the next few weeks to receive word of a follow-on Phase II grant that would fund the project over the next few years.

However, KGI and Claremont still work together very closely, with Claremont potentially serving as the commercial conduit for any technologies developed at KGI.

"The amplification and detection for TB is coming from [KGI's] side," Doebler said. "The lateral flow is coming from our side, and it integrates into the cartridge; and [KGI] has an amplification and detection technology, and we're integrating that."

Claremont's proposed C. difficile assay has used "a number of different isothermal amplification reactions, very similar to what Angelika's group is using." He added that the company was recently awarded a patent for a miniature fluorimeter that is "geared toward isothermal nucleic acid amplification and keeping the temperature very [stable]. So for C. diff we have our own platform that we're integrating, which will be fluorescence-based instead of lateral flow-based. We've sort of got our hands both projects."

Doebler added that even the sample-prep cartridge for each integrated device is currently slightly different. "The TB cartridge will be a little different from C. diff, because we're dealing with sputum versus stool," he said. "There is probably a common ground where we could come in with a common cartridge that could work for a number of bugs."

Doebler also said that it was too early to comment on potential commercialization timelines, noting that much depends on continued funding of the company's C. difficile project. However, he noted that Claremont "is always talking with companies about the potential integration of our sample-prep technology with their detection technology. Those discussions are ongoing. We've had some interest with companies that maybe don't have a C. diff assay yet and are competing with some of the large companies that do."

Meantime, Niemz noted that her group at KGI hopes to demonstrate integrated functionality of at least its TB device by next summer, and has designs on beginning a small clinical study by the summer of 2014.