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UMDNJ, Cepheid Win $1.3M for Blood-Based Biothreat Tests; Cepheid to Use Tech in MDx Products

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By Ben Butkus

Researchers from Cepheid and the University of Medicine and Dentistry of New Jersey have won nearly $1.3 million from the National Institutes of Health for the first year of a five-year project to develop sample prep methods and nested PCR assays to detect bloodstream infections, PCR Insider has learned.

The overarching goal of the grant, which is being administered by the National Institute of Allergy and Infectious Diseases through its Partnerships for Biodefense special emphasis program, is to develop cartridge-based tests for Cepheid's GeneXpert system that could detect a variety of bacterial bioterrorism agents and be rapidly deployed in the case of a terrorist attack.

However, Cepheid expects that the blood-based sample prep methods its scientists are developing under the grant will find use in future molecular diagnostic products to detect myriad infectious agents from a wide variety of difficult sample types, David Persing, Cepheid's chief medical and technology officer, told PCR Insider this week.

"We want to … maximize our sensitivity for detecting any bacterial agent that happens to be circulating in whole blood in the context of a sepsis evaluation by using select [biothreat] agents as a proof of principle, and maximizing the performance of our sample preparation," Persing said.

"We expect to be able to leverage that for multiple products in the future — whether it's for sepsis, ventilator pneumonia, urinary tract infections, or other infections," Persing added. "We expect to be able to use similar approaches to get into direct specimen detection, by concentrating organisms from large volumes of blood, alveolar lavage fluid, urine, or other samples."

The project began last week and is scheduled to continue until February 2017. Principal investigator on the grant is David Alland, a researcher at UMDNJ and a long-time Cepheid collaborator. Alland told PCR Insider this week that the requested budget is about $5.5 million over five years.

"This grant is to support work to develop an assay to detect select agents of bioterrorism directly from patients' blood," Alland said. "It uses the Cepheid [GeneXpert] platform, which is a very flexible platform for designing assays. And one of the innovations of this project is that it lets us design assays quickly and that can be used on the basic platform to get them out rapidly when needed."

In a recently published abstract describing the project, the researchers noted that most bacterial bioterrorism agents cause bloodstream infections, and that blood-based detection of these agents offers the promise of early diagnosis.

However, rapid detection of bloodstream infections is particularly challenging because the assays must be extremely sensitive — on the order of five colony-forming units per milliliter of blood — yet must also be rapid, robust, and easy to perform, preferably at the point of care, the researchers wrote.

"Even the perfect select agent test will be of little use if it is not available when needed in an emergency," the abstract states. "Thus, the best select agent assays must be able to be manufactured upon very short notice, or be very inexpensive, easy to stockpile, and have a prolonged shelf life. The very best assays would have all of these characteristics."

To this end, Alland and colleagues are exploring use of an approach that they recently discovered to automatically extract bacteria from one to 10 milliliters of blood and then perform "very-small-amplicon nested PCR" in a disposable cartridge on the GeneXpert system.

"What's nice about Cepheid's platform is that it's very flexible, and it allows you to design different assays with your desired characteristics, and you can do nested PCR inside the platform, which is a very sensitive, specific assay," Alland said. "And you can develop assays relatively inexpensively, because you don't have to develop a new platform every time."

The grant abstract did not provide details about the technological approach that the researchers are employing; however, a proof-of-principle study published by Alland and others last month in PLoS One provides some specifics.

In their study, the scientists developed GeneXpert-based nested PCR assays for Staphylococcus aureus and used them to test blood samples from patients with bloodstream infections.

According to the paper, the team processed the blood samples in GeneXpert cartridges containing a special filter to capture intact bacteria. "Different chambers within empty [filter-based] cartridges were manually filled with PCR reaction mix or sample-processing buffers," the researchers wrote. "A blood sample was added to a [filter-based] cartridge's sample chamber, and the cartridge was then inserted into a GeneXpert system module. The sample was then processed within the cartridge by the GeneXpert system according to a specified protocol using customized fluidics software."

The bacteria were then lysed by agitating glass beads within the filter chamber using an ultrasonic horn, and the liberated DNA was then washed through the filter into a collection chamber for further on-cartridge nested PCR.

Using this protocol, Alland and colleagues were able to detect S. aureus in about 90 percent of patient samples. More importantly, the assays took about two hours each to complete and required just 1 milliliter of whole blood.

Using the NIAID grant, the researchers will now attempt to adapt their protocol for rapid point-of-care assays on the GeneXpert. According to the grant, specific aims of the project include developing highly sensitive and multiplexed nested PCR assays for several category A and category B agents as defined by the US Centers for Disease Control and Prevention; adapting each assay to the GeneXpert platform; optimizing the protocol to isolate select agent DNA from 10 ml of blood using Cepheid's cartridges; and "developing an emergency plan for on-demand manufacture" and emergency use authorization from the US Food and Drug Administration.

MDx, Not Biodefense

Cepheid already plays in the biodefense market, as it has a longstanding collaboration with Northrop Grumman and the US Postal Service to provide post offices with anthrax detection tests on the GeneXpert system. To date, the company has provided more than 10 million assays for this purpose; yet it does not foresee leveraging technologies developed under the NIAID grant for this purpose.

"Strategically, biothreat detection is not an area that we are going to be focusing on as a company because … the need for biothreat detection is often related to both the political environment and the perception of threat, not so much the actual threat," Persing said. "So there are a lot of variables and things that we can't control that dictate the supply-and-demand equation, and that's not an ideal situation as a company."

Instead, Cepheid plans to incorporate some of the technologies — specifically the sample prep components — in future molecular diagnostic products.

"The bottom line is, what we expect to see happen with this program are some important proof-of-principle experiments using biothreat agents as a testing methodology," Persing said. "The biothreat select agent work will be done in Dr. Alland's lab … using open cartridges. He will actually be developing the reagents for biothreat detection and testing them in his laboratory."

Meantime, Sunnyvale-Calif.-based Cepheid scientists, some of whom will be directly supported under the NIAID grant, will focus on the sample prep aspects, particularly from whole blood.

"Whole blood is one of the most challenging specimens in all of clinical diagnostics for sepsis detection … and capturing and concentrating those organisms and getting all their DNA into a small, 25-µL PCR reaction is probably the biggest challenge of any company getting into the sepsis detection space," Persing said.

"So we're going to be focusing on ways to do just that — take the GeneXpert cartridge technology … and show that what it really does well is to concentrate specimens from large volumes," he added. "The goal would be to develop a system that could be used as a rapid front-line diagnostic system directly from the specimen without needing prior culturing."

Cepheid already markets GeneXpert tests for detecting S. aureus, methicillin-resistant S. aureus, and other sepsis-causing organisms from blood culture bottles, and the tests have been well adopted. However, for these types of assays "it is not often understood that you've got to wait for 24 to 48 hours before those cultures become positive, so there is a significant delay between getting that sepsis result and getting the organisms identified," Persing said.

"Ideally, clinicians — especially [intensive care unit] docs — would like a rapid result that identifies organisms down to the species level, and ideally includes drug-resistance targets, either in the same system or on a reflex basis, that would enable them to [prescribe] better-informed therapies," he added.

Cepheid has previously reaped the benefits of collaborative NIH grants with Alland's lab. In 2009, Cepheid and UMDNJ received a $7.5 million grant from NIAID to further develop the multiplexed detection capabilities of the GeneXpert system in order to create a new assay for detecting multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis.

The performance of that TB test has since been vetted in multiple studies, and Cepheid now markets it primarily in both developing countries and more industrialized nations (PCR Insider, 9/2/2010).

"This is very similar in many ways to the program we did on tuberculosis with NIAID, where they funded cartridge technology development that was very much tied into what David Alland was doing on his end with molecular beacon assays and TB," Persing said. "We considered it to be a win-win, because now many of our other assays use that six-color detection technology, and it was a great benefit to the company to have at least part of that expense covered. We expect to see similar benefits — direct and indirect — as a function of this program."


Have topics you'd like to see covered in PCR Insider? Contact the editor at bbutkus [at] genomeweb [.] com.

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