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PositiveID Developing Handheld PCR for Space, Biodefense with Eye Toward Clinical Apps


NEW YORK (GenomeWeb) — Biodetection and biodefense company PositiveID recently announced a number of research awards and partnerships, including with the US Defense Threat Reduction Agency and NASA, which it is using to develop its handheld microfluidic PCR device, called Firefly Dx, for government uses.

However, the Delray Beach, Fla.-based company is also looking to leverage these opportunities to expand into commercial markets such as clinical testing and agriculture, ideally with a partnering organization, Lyle Probst, PositiveID's recently appointed president, told PCR Insider in an interview last week.

As reported in PCR Insider, last year PostiveID began developing a larger PCR platform, called Dragonfly, into a cassette-based, battery-operated diagnostic instrument, citing a burgeoning market for handheld devices.

This market also now includes platforms for PCR in space. A recent NASA conference paper described a 2011 Johnson Space Center workshop on microbial monitoring for long-term missions, which recommended molecular-based methodologies such as PCR over current culture-based methods.

This kicked off a multi-center collaborative research effort "to explore novel commercial-off-the-shelf hardware options for space flight environmental monitoring." The platforms had to be closed systems requiring minimal reagent mixing, and easily modifiable for microgravity. Selected commercial PCR platforms were then challenged to detect low levels of Salmonella enterica.

Two platforms were ultimately recommended from this head-to-head comparison — Razor Ex by BioFire Diagnostics (recently acquired by BioMérieux), and a platform called iCubate, previously described by PCR Insider. A third platform, Cepheid's SmartCycler, was deemed unsuitable, as it was considered labor intensive and did not provide enough sample containment for microgravity.

The study, however, concluded that "future work will include ... identify[ing] any additional current technologies that meet the needs for space flight application."

To this end, FireflyDx may provide a more optimal solution than a benchtop platform. Probst explained that NASA has now partnered with PositiveID to develop its device for three different types of use.

The first is for Mars missions, to automatically collect soil samples and analyze them for signs of life. The company is currently testing soil samples provided by NASA that are "representative of what they expect on Mars," Probst said. They do not expect to detect organisms in these, but will develop the sample prep methods, and also spike in organisms.

The second use integrates a NASA-developed multiplex PCR-based technology for detecting up to 30 organisms simultaneously, Probst said. This is important to healthy space travel. NASA recently described a method in Applied Microbiology and Biotechnology to sample the microbiome on the International Space Station. Because some organisms are present in higher numbers, and PCR amplification steps skew the census away from lowly-expressed but potentially dangerous pathogens, the new technique uses propidium monoazide treatment coupled with downstream qPCR and pyrosequencing.

This area of Firefly development also includes a blood-based biodosimetry test to measure how much of the radiation bombarding astronauts has actually entered an individual person.

"With [the NASA] capability on the cartridge, it could be used to look at infectious diseases in humans, whether it's here on Earth or in space. It could be used to assess the health of astronauts, not only in space flight but also on the International Space Station," Probst said.

The third domain of collaboration is atmospheric monitoring, with a special focus on the fungal spores that cause Valley Fever. "Part of what NASA is looking to do is monitor the atmosphere for signs of this spore, to look for the movement of it, and see if it shows up in new regions," Probst said. The application would involve taking Firefly cartridges up into tethered balloons or unmanned aircraft, pulling and processing samples, and reporting the results back to Earth.

NASA is also partnering with other commercial entities through the Space Act Agreement, Probst said. However, he added, "under our agreement with [NASA], if there is a capability that another corporation has that they're looking to integrate or use, then we can work in a collaborative environment with them, as opposed to compete for that work."

PostiveID's well-established methods and lab-equivalent results for fieldable tests appealed to NASA, as did running time and projected cost of the tests, Probst said. The low cost — somewhere between $15 and $30 per test — and the fact that it required no special training also enables low-resource testing, he said.

The assay-specific cartridges have four-color detection capabilities and use around 30-microliter reaction volumes. The current configuration runs 40 PCR cycles, and takes less than 15 minutes.

The speed and portability are not due to chemistry differences, Probst said, but to the proprietary hardware. The device was, in fact, designed to use standard PCR chemistries so that the end user will not need additional training to read results. Concentrations, volumes, and enzymes are also standard.

"If an end user already has an assay that they like, if it's a standard TaqMan assay, we can port it onto the system without changing the chemistry," Probst said. "We don't have to go out and create a new assay for every organism that anybody wants to look at."

The cartridge uses lyophilized PCR reagents, which are stable at room temperature for long periods of time. The reconstitution of the reagents happens from the sample processing, Probst said. Sample is injected into the cartridge at volumes of between 50 and 250 microliters. The cartridge also uses stored liquid reagents in sample purification. The resulting pure sample suspended in liquid reconstitutes the lyophilized reagents.

Along with the NASA agreement, PositiveID and partner Leidos were recently awarded a contract from the US Defense Threat Reduction Agency to develop detection technologies for potential weapons of mass destruction.

This DTRA award is, in part, for development of PositiveID's microfluidics-based Bioagent Networked Detector, or M-BAND, technology, and could be worth up to $4 billion.

In contrast to the handheld Firefly device, M-BAND is "about the size of a refrigerator," he said. "It is that large because it was designed to ... pull in air samples and monitor for biothreat organisms in an autonomous fashion." The device performs sample prep and PCR from air samples. It can provide PCR results every three hours, 24 hours a day, for 30 days, before it needs its reagents restocked.

"It's a lab in a box," Probst said.

It is also designed to function outdoors, and can withstand large external temperature fluctuations without compromising the PCR happening within. To do this, the enclosure has a large air conditioner on one side, Probst said.

In conjunction with the Department of Homeland Security, M-BAND was field tested in Boston for one year. "We had units at the Seaport, in the Metro, and on rooftops," Probst said. They also tested ten devices in London. These tests are now complete, and did not find any biothreat organisms. Chamber tests, in which the devices were exposed to DNA from dangerous organisms, have also been done.

Under a Department of Defense program called Jupiter, PostitiveID is now sending M-BAND units to the Aberdeen Proving Grounds and to Korea. This is a test and evaluation, not a procurement program, Probst said. The program evaluates M-BAND for future use by military organizations. When it is finished, "they have an instrument that's been evaluated by the Department of Defense that they can procure and deploy without having to go through an evaluation first."

"The majority of our work to date has been under government contracts," Probst said, primarily in the biodefense field. Fielding M-BAND, for example, was enabled by a $35 million contract over seven years.

In addition to government contracts, however, the company is now diversifying to commercial applications with the technology already developed. "Firefly is the first instrument that we're putting through that process. It has government applications, but also applications in clinical and agricultural settings. That's where our commercial efforts have picked up, in these other diagnostic and detection markets."

In terms of the clinical market, Probst said the Firefly system was designed from the beginning to be able to achieve a CLIA waiver. The company also plans to go through the US Food and Drug and Administration process. "That is a second or third phase for us," he said. "There are other markets where [a lengthy approval process] is not required, that we can put the Firefly into earlier. The information generated in these other markets and applications can be used in the FDA evaluation, so it will help streamline the FDA process," said Probst.

He also said that this expansion makes sense given the company's roots.

"Whether you're looking at biodefense, clinical, or agricultural applications, you still have to be able to pull in a sample, purify out your nucleic acid, and run PCR," he said. "The samples that we've processed in the biodefense area, the environmental samples, are some of the most difficult ones to be able to purify nucleic acids out of, and we've been very successful at it. When we started looking at clinical samples - blood, saliva, sputum, urine — those were actually very easy to process compared to what we had been dealing with."

PositiveID is now actively looking for partners. "Our objective is not to be a newcomer to break into these markets, but to partner with the appropriate companies already in these markets and bring a new technology," Probst said.

There are prototypes of Firefly Dx, and some existing data, but the company is also seeking investment for further development, in the form of a partner or possible end customer who would like the device tailored specifically for its needs.

M-BAND, however, is currently licensed to Boeing for domestic and government use. "We work very closely with them to manufacture and run it. They are more interested in the overall network system, [since] it has to fit into a network for government needs." Under this agreement, PositiveID maintains exclusive rights to supply reagents for M-BAND.