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PATH Team Completes Prototype Electricity-free Reusable MDx Platform; Eyes Disposable Version


NEW YORK (GenomeWeb) — Scientists from the non-profit research institute PATH have published a paper describing their non-instrumented, electricity-free, reusable point-of-care molecular diagnostic platform, and demonstrated its ability to sensitively and reproducibly detect HIV-1 from human plasma samples in less than 80 minutes.

According to the researchers, the platform — called NINA for non-instrumented nucleic acid amplification — is designed to accommodate a wide range of isothermal amplification methods, and could be tailored for the detection of a variety of infectious diseases. As such, the team is currently seeking external partners to bring the technology to market.

In the meantime, the team is already pushing ahead with a disposable version of the NINA technology that could be even more amenable to point-of-care use, especially in resource-poor areas of the world, Paul LaBarre, a senior technical officer at PATH and principal investigator on the project, told GenomeWeb in an interview.

The new paper, published in November in PLOS One, "focuses on one configuration of [NINA] that is reusable," LaBarre said. "As long as you can reload it with new fuel and initiate the exothermal reaction with saline … you can use that fairly indefinitely, hundreds of times.

"But our new direction … [which] we're going to push to publish as quickly as possible, is this disposable option, which is smaller, and made out of materials … where you're really not throwing that much away," he added. "The big highlight is that you could put this into a backpack, go into a village with absolutely no electricity, and get excellent results within a half hour or hour. And that's super-exciting to us."

The researchers first presented the concept of NINA in a 2011 PLOS One paper. There, they demonstrated proof of concept for incubating isothermal nucleic acid amplification assays using an exothermal chemical reaction of calcium oxide and water combined with an engineered phase change material.

A few months later, LaBarre and colleagues received a grant from the National Institute of Biomedical Imaging and Engineering to develop NINA into a kit to diagnose diseases in resource-poor areas of the world. Under that grant, the PATH researchers forged a partnership with the US Centers for Disease Control and Prevention specifically aimed at using NINA with reverse-transcription loop-mediated isothermal amplification (RT-LAMP) to diagnose HIV-1.

Since that time, the collaborators have greatly improved and streamlined such an assay, work that is described in last month's PLOS One paper. For instance, instead of the exothermic CaO reaction combined with a phase change material, which "achieved acceptable performance but exhibited considerable variability," the researchers adopted a magnesium iron alloy, MgFe.

The advantages of this, they explained, include the fact that MgFe has a significantly higher energy density, reducing the mass of the fuel pouch from 20 g to 1 g; is commercially available at a very low cost and with little batch-to-batch variation; and can be milled to a specific particle-size range to further control the heat profile of the chemical reaction.

Other improvements to the NINA design included "packaging the MgFe fuel in a hydrophilic, heat-sealable membrane and containing the liquid reactant in an easy-to-use blow-fill-seal container," thus greatly minimizing user steps and eliminating post-amplification heater cleaning; and incorporating a "smaller vacuum-insulated housing to reduce heat loss and decrease the overall size of the heater," the researchers wrote in their paper.

Finally, the team incorporated an improved phase change material with very high latent heat to meet the thermal requirements of an HIV-1 LAMP assay; and incorporated a nucleic acid lateral flow visual detection method for easy assay readout.

LaBarre and colleagues demonstrated the robustness of their platform by evaluating its thermal performance over a wide ambient temperature range representative of low-resource settings. Specifically, they evaluated nine NINA heaters over 74 individual one-hour runs, and were able to demonstrate highly precise temperature control with one standard deviation between 60° C and 62° C — the ideal temperature range for a LAMP assay — except in a few cases where the device underwent mechanical failure due to specific elements of its fabrication.

The researchers then evaluated the performance of a biplexed LAMP assay for the detection of HIV-1 and a β-actin internal control running on either the NINA platform or a commercial real-time PCR system, and found that results using either heating method were similar, with a limit of detection of 75 copies per reaction or 8,333 viral copies per milliliter of extracted plasma.

The PATH researchers noted in their paper that further optimization of the biplex LAMP reaction is expected to improve the sensitivity of the assay. To wit, they have since adopted a LAMP assay developed by their CDC collaborators. "[CDC has] a very solid LAMP assay they are advancing right now and have gotten excellent results on," LaBarre said. "So on the science side, I think they've exceeded our performance specifications on the LAMP assay. But I think we've completed the engineering on the reusable configuration, and we're looking for a commercialization partner on that."

Although the prototype NINA assay for HIV-1 uses LAMP as the isothermal amplification chemistry, LaBarre said that the intent is for the platform to be usable with "pretty much any isothermal method" including recombinase polymerase amplification, cross priming amplification, and helicase-dependent amplification.

"Each isothermal method might have a slight variation on the device because it might have a different optimal temperature that it operates at, but that's something that we can easily customize to and have a 65 degree [Celsius] platform that's good for CPA, HDA, and LAMP because they all share the same polymerase; and then another one that's good for RPA, at a lower temperature."

Fully disposable

Even as the LAMP team seeks commercialization partners for its reusable configuration of NINA, it has begun work on a version in which the entire incubation device would be fully disposable. Such a device, LaBarre posited, could achieve a CLIA waiver and thus be widely disseminated not only in the developing world, but in more industrialized regions where a true point-of-care infectious disease assay would still be a great boon.

"I could even see these on the shelf at Rite Aid, where you open up the kit contents, and you have everything you need to draw the sample and conduct sample prep, [although] we're looking to get rid of all sample prep through the use of very robust enzymes," LaBarre said. "And then the amplification heat reaction is initiated by, say, shutting the door on the device. And then through another external manipulation, after the reaction is done, you initiate flow of your amplified product onto a lateral flow strip, and then it's a direct readout."

Such a device might end up costing a bit more than the reusable configuration, which figures to have a low one-time cost associated with the device itself, plus the price of each assay, the raw materials for which are estimated to cost around $.06 per reaction. This does not include any costs that would be built into the assay due to licensing fees for an isothermal amplification reaction.

"But I think the disposability gets you the trade-offs of convenience and safe biohazard disposal without having to clean the device, which is nice when considering something like Ebola," LaBarre said. "So it probably comes with a small per-test cost increase as opposed to a reusable configuration."

In addition, "we're still working to get the same performance on the disposable configuration as we can get on the reusable, so there might be a slight drop in performance, at least until we optimize it."

Should PATH successfully develop a working prototype disposable NINA assay, LaBarre envisions various commercialization partners selling it as a kit for specific diseases. At that point, the researchers would need to decide on a specific isothermal amplification technology for each new kit developed.

"We are definitely not committing to any one of them, and from a platform licensing standpoint for PATH … we would be looking at non-exclusive licensing so that if the best product for Ebola, for example, is HDA, then we go for that," he said. "But if the best product for HIV early infant diagnosis is LAMP, we want to make sure we have that opportunity, as well, by not extending any exclusivity."

The NIBIB grant on which LaBarre serves as principal investigator is set to run through August of 2015, through which time the PATH researchers will continue to work with CDC on the HIV-1 assay as well as tests for other diseases.

Coincidentally, another research team at PATH led by David Boyle is currently working under a National Institutes of Health grant awarded at nearly the same time as the LaBarre group's grant to develop a minimally instrumented point-of-care nucleic acid amplification test using RPA to diagnose HIV-1 in infants in resource-poor areas of the world. Last year that team, along with collaborators from the Fred Hutchinson Cancer Research Center and Alere subsidiary TwistDx (which owns the RPA method), demonstrated that the assay could quickly and accurately detect a number of strains of HIV-1.

Boyle's team "works very closely with our team here, and we really step into each other's projects and publish together," LeBarre said. "Our project is an engineering platform kind of conversation, where we've identified an overall broad need for … a simplified electricity-free process; whereas Dave Boyle has more of an expertise in HIV, polio, and [tuberculosis], and is interested in finding a direct solution to a specific disease problem.

"We're more interested in further demonstration of a platform that could be used across multiple diseases," LeBarre added.

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