This article has been updated from a previous version to correct the names of an individual and research institution.
The point-of-care molecular diagnostics market is "a race to the bottom" in terms of cost and turnaround time, and it is being fueled by rapid innovation in microfluidics, miniaturization, and nucleic acid amplification, detection, and sequencing technologies, according to industry insiders.
However, according to some experts, many of the molecular analysis tools needed to enable reliable POC molecular diagnostics are already in place, and some of the biggest remaining hurdles are peripheral components such as reagent storage, sample prep, and integration with existing medical infrastructure and electronic record keeping.
And, according to at least two early-stage test developers, the commercial market is still hamstrung by the fact that the inherently low cost of POC molecular tests means that a relatively large amount of product needs to be sold immediately to justify investment — a tipping point that is difficult to achieve for most of the smaller companies and academic labs responsible for innovation in this area if they do not have help from larger development partners.
POC diagnostics based primarily on immunoassay technology have been around for decades. Similarly, molecular diagnostics — particularly PCR-based nucleic acid analysis — have largely replaced immunoassays in centralized clinical diagnostic labs and hospitals due to their superior accuracy and sensitivity, and their ability to be at least semi-quantitative.
However, molecular tests are not nearly as easy to implement in non-laboratory settings as immunoassays are —mostly because molecular tests have traditionally required complex, bulky, and expensive instrumentation. As such, the market for POC molecular diagnostics has been slow to develop — although a market certainly exists, both in resource-poor and more industrialized areas of the world.
"To me, POC really is if you test someone, you should be able to treat them at the time of diagnosis, while they're actually there," said David Boyle, a senior research scientist at the Program for Appropriate Technology in Health, or PATH, a Seattle-based non-profit global health organization and research institute.
"This is a big problem with mother-to-infant transmission of HIV [in the developing world]," Boyle added. "The mom turns up with the baby at a maternal wellness [or] pre-natal prevention [clinic] and they take a sample, and that gets shipped off to a big lab and the results get sent back. The number of people that don't come back to get that test result is still really high."
Boyle and his colleagues at PATH, Alere subsidiary TwistDx, and the Fred Hutchinson Cancer Research Center are attempting to develop a non-instrumented molecular diagnostic test to diagnose a variety of diseases — particularly HIV-1 — at the point of care in the developing world (PCR Insider, 8/25/2011).
The team recently completed a study showing that the assay — which uses TwistDx's isothermal recombinase polymerase amplification, or RPA, technology — can sensitively detect and differentiate HIV subtypes using either lateral flow strip or simple fluorescent readout detection in about 20 minutes, data that will soon be published in a peer-reviewed journal.
"We may know the mom has HIV … and she's being screened to ensure her infant hasn't contracted it," Boyle said. "We're thinking that if we had a test … where they get a result [immediately] rather than not knowing or not going back [due to] cultural and financial issues, there is more opportunity for the infant to go on [anti-retroviral therapy]."
The potential market for such a test extends beyond resource-poor areas of the world and onto the battlefield, according to Boyle.
"The military is always really interested in this kind of stuff — in a lot of places where soldiers have to go fight, they're exposed to a lot of nasty diseases," he said. "Again, it's the same thing — you want something that's small, easy to use, highly accurate, and gives a result really quickly."
Most of the POC molecular diagnostics being developed for resource-poor areas of the world or the battlefield aim to diagnose infectious disease. However, in industrialized regions, POC molecular tests also have tremendous value potential, according to Keith Batchelder, CEO of personalized medicine consultancy Genomic Healthcare Strategies.
"POC molecular diagnostics [are] heating up … certainly in infectious disease areas … but I think you will see POC diagnostics used in multiple settings, both the developing world and industrialized world," said Batchelder, who chaired the POC diagnostics track at Cambridge Healthtech Institute's Molecular Medicine Tri-Conference in San Francisco this month.
"The industrialized world is looking for POC diagnostics to be able to move care into lower-cost settings," Batchelder said. "[For example], if you have a quantitative molecular diagnostic that allows a nurse practitioner to diagnose and appropriately treat a patient … that saves money for somebody. Who that somebody is — that depends."
One such 'somebody,' he noted, will likely be the pharmaceutical industry, especially as companion diagnostics become more prevalent.
"As we see companion diagnostics being required for new therapeutics, I think pharma would love to have point-of-care tests," Batchelder said. "The perfect point-of-care molecular diagnostic for pharma would be free; distributed widely; usable by the [patient]; and tightly coupled to their therapeutic and not any other therapeutic. Whether or not those things exist is a different story, but pharma is certainly looking for them."
A molecular diagnostics firm that has taken a step in that direction is Ottawa, Ontario-based Spartan Bioscience, whose point-of-care CYP2C19 test to identify patients carrying a mutation associated with poor response to anti-platelet therapy following cardiac stent insertion is currently commercially available in countries recognizing the CE mark, and is awaiting marketing approval from the US Food and Drug Administration (PCR Insider, 11/17/2011).
A number of CYP2C19 mutation molecular tests are available through centralized laboratories, but the threat of major cardiac events in heart attack patients within the first 24 hours to 48 hours of receiving the anticoagulant drug Plavix has upped the need for a point-of-care test with a fast turnaround time.
Spartan's test runs on the Spartan RX, a fully automated, low-cost, portable, endpoint PCR-based DNA testing device. The company has staked its claim as one of the first providers of true sample-to-answer point-of-care molecular testing, but is already looking to the future and how it can make its testing platform even more conducive to point-of-care use.
According to Paul Lem, founder and CEO of Spartan, molecular testing is slowly following in the footsteps of lateral flow immunoassay testing.
"In the early days, all these immunoassays were lab-based, and then it started getting down to POC, and the first ones were really bulky and expensive, but, over time, everyone has been refining it to the point where it's touch-screen, it interfaces with [laboratory information management systems], takes a few seconds to run, the cartridges cost $1 or $2, and it's easy enough to be used by anyone."
Spartan's platform is currently "about the size of a shoebox," and as such is not nearly as "point of care" as a lateral flow immunoassay. However, "our first generation does everything that we want it to do, but we already see the path that's been laid out for us by the lateral flow [market]," Lem said. "Over the years we will keep iterating — the next thing we are thinking about is shrinking it down from the size of a shoebox to a handheld device. Then we're thinking about shrinking the time down from about 60 minutes to around 5, 10, or 15 minutes. And then, of course, get the cost down."
In general, it is inevitable that the cost of POC molecular diagnostics will drop, said Genomic Health Strategies' Batchelder.
"Multiple economic drivers are increasing the attention being paid to POC," Batchelder said. "The interesting thing about POC diagnostics is that they are benefitting from the race to the bottom of all of the sequencing technologies; microfluidics technologies; all the people who have been devoting time to miniaturization."
Another trend that Batchelder sees is the integration of molecular testing with technological innovations from other industries, such as information technology and mobile communications.
"There are now network devices, that can be used to manage the [diagnostic] information and connectivity, and that seem an awful lot like an iPhone," Batchelder said. "I'm not suggesting that everything is going to be iPhone attached. I'm merely suggesting that there is a whole confluence of network connectivity, electronics, and sharing electronic patient information that's happening that makes POC diagnostics more feasible. It's now not unreasonable to think of a POC diagnostic at home, attached to something [via] Bluetooth that then communicates to something else."
Spartan's Lem echoed this idea, noting that his company has also been exploring ways to wirelessly connect its molecular testing platform with some sort of central mainframe so that results can be reported immediately to physicians even if they aren’t present at the point of care. One attractive way to do this is to take advantage of existing smartphone technology.
"We're looking at that sort of thing — a smartphone connected to a handheld DNA analyzer," Lem said. "Smartphones are becoming ubiquitous; they have such volume that prices have come down so much; and it's like a supercomputer in your pocket. I think we'll start seeing more devices that use smartphones as their data interface, and for connectivity, data analysis, all those things. Then you can imagine … data that feeds [directly] into your medical records."
At least one research group, led by Michigan State University professor Syed Hashsham, is already exploring commercialization routes for a handheld nucleic acid testing device empowered at least in part by smartphone technology.
The Hashsham group's device, called Gene-Z, started out several years ago as a handheld nucleic acid analysis platform for identifying pathogens in water. But over the past few years, the group has developed a prototype battery-operated device that uses microfluidic consumable chips to detect DNA, RNA, and microRNAs in 10 minutes to 30 minutes; interfaces with an iPhone or Android phone for data analysis and connectivity; and could cost less than $1,000 with consumable chips costing between $2 and $20 (PCR Insider, 3/22/2012).
The device also started out capable of accommodating anywhere from 64 to 1,536 1-mL wells, but Hashsham said recently that, after getting feedback from physicians, the group has simplified its testing chips even more.
"From physicians and other people we talked to, it was obvious that something with a smaller capacity and even lower cost would be very useful," Hashsham said. "The previous version had 64 wells, and they are now eight to 12 wells, more like a single assay system that could be done by anybody provided they had the chip."
Although Gene-Z is still in prototype stage, Hashsham said his group is seeing interest from "wherever we can answer the questions: 'Why should we use it? What is it changing for me?'" In many cases, he said, the answer is that the device, even if it costs the same as laboratory-based molecular testing, is saving time and effort because an answer can be obtained nearly instantly.
"And in some cases, it is saying that these tests were never possible, and mostly that's coming from the human genetics side, and there are many," Hashsham said. "Many [human genetic tests] are so complex that they will never be a POC type of test. But there are some where you could. If you're talking about a single mutation — Factor V Leiden [mutations], for example, [for hypercoagulability disorder] — that's a test that's already done in hospitals, and if it is something that I [can diagnose] very easily, it will be beneficial. And the test is not that complicated."
As is the case with many groups developing point-of-care molecular diagnostics — and even centralized molecular diagnostics — sample prep remains a formidable hurdle for Hashsham and colleagues.
"If you're going to be talking like it's going to be used in the field, you cannot really avoid that," he said. "[For example] in a hospital setting, saying we are going to use it with blood [samples], we almost have to show that we are doing something to the blood or plasma or serum, some sample concentration."
"We are addressing this, and basically at this point is you can't avoid sample processing in every occasion, but … we are saying 'Let's wait and ask [whether] we need it, and go ahead and find the applications and samples where we don't need it. There will be some samples where you need [nucleic acid] concentration, if not separation. We are trying to avoid lysis and DNA extraction, and if you can avoid it completely, then that is advantageous for point of care testing."
Spartan's Lem said that the most difficult part of the Spartan RX and CYP2C19 test was "that front end — collecting the sample, extracting the [nucleic acid], and having it PCR-ready every single time. It's kind of like a 99 percent to 100 percent reliable bar — it has to be that good to make clinical grade, and to get molecular [testing] to that point is really difficult. Lots of people have their proof of principle in a laboratory, and it works like 75 or 80 percent of the time, in ideal conditions. But to get it out in the real world … that's really difficult."
The PATH team and others developing POC molecular diagnostics for the developing world may have an even greater challenge, considering the often harsh environmental conditions in those regions. Boyle said that the RPA reagents that TwistDx manufactures for the PATH HIV test are shipped in a lyophilized form, and initial observations seem to indicate that the reagents hold up under challenging weather conditions.
Nevertheless, as part of its development efforts, the PATH group is now undertaking a study to measure the tolerance of its assay reagents to different environmental conditions.
"They come in sealed foil pouches, so we're not bothered about humidity," Boyle said. "But we'll be doing stability exercises, reactions held at 30, 35, and probably 45 degrees Celsius, and just doing a timed experiment, three or four months, to see how long these materials can stand being at that particular temperature."
Boyle noted that this is an important undertaking because the majority of molecular diagnostic manufacturers only guarantee reagent stability up to about 30°C. "At PATH we've found that … [in some of these areas] it's not so much that there is one temperature, it's that there are gross temperature fluctuations," Boyle said. "Is 30 degrees Celsius an acceptable upper temperature to look at stability? Because in a lot of places the ambient temperature is actually higher than that. I don’t know the answer to this, but why is 30 the magic number?"
"Ideally, what we would like is if they could be thermostable at 45 degrees for three months, and stable for a year at 35 — that would be a lot more useful … if something is in a truck or stuck in a shed for a while," he added.
Another topic up for debate in the POC molecular diagnostics world is which molecular detection technology is ideal. PCR and real-time PCR have been the technologies of choice for centralized molecular diagnostics for a decade or more, and they also are the most obvious candidate for POC testing. However, because those technologies still require at least some kind of instrumentation, they may not be the best for extreme POC tests like the one the PATH is developing.
Still, according to Spartan's Lem, PCR remains the king.
"The market has voted," he said. "Over the last 10 or 15 years all these technologies have tried to compete with real-time PCR: isothermal amplification, [nucleic acid sequence-based amplification], [peptide nucleic acids], label-free — every possible variation. And it's shaken out that PCR is dominant. It has some really attractive properties, which is why it's dominated in the lab and why I think it will dominate in the POC. Those properties would be that it's really sensitive and specific … and in the real-world setting it's been shown that it works the best in clinical applications."
Another advantage, Lem said, is that the infrastructure for real-time PCR is already in place, such that "you can order the probes, the primers, from so many different shops; whereas it's hard to order the enzymes even for something like isothermal. There are just way fewer distributors."
Hashsham's group at MSU has taken the same tack. Whereas Gene-Z originally used a proprietary quantitative isothermal amplification method, Hashsham said recently that the team is now integrating real-time PCR capabilities into the system.
"There are many SNP-based tests that are much more difficult by isothermal amplification, at least in terms of the primer design," Hashsham said.
But from where Genomic Healthcare Strategies' Batchelder is sitting, the technology choice doesn't matter as much, at least to end users — so long as the test provides value.
"First off, the old lateral flow diagnostic is still doing pretty well," he said. "Second, I am very focused on what content and value proposition is going to drive a POC diagnostic to be successful." For instance, he noted, "CD4 counting in Africa is … very expensive to do it any other way than in a cheap POC diagnostic instrument. That's a value proposition, so I don't really care what the technology is." Picking which molecular testing technology will win the day in POC testing "sounds to me like a stock picker's job," he added.
Ultimately, the most daunting aspect of developing POC molecular diagnostics may be that no one wants to invest in their development. Tying a diagnostic to a specific pharmaceutical for personalized medicine has certainly attracted funding from both pharmaceutical firms and large diagnostic developers, but gene tests without such a clear-cut therapeutic link, or tests for infectious diseases, may have a longer path to travel — at least according to Boyle and Hashsham.
"We have to get more funding," Boyle said. "We've still got a year and a quarter of [an National Institutes of Health] grant, but with funding the way it is, and given that it takes about nine months to review a grant … we're already thinking about who might be interested in funding this more."
Boyle said that the Bill and Melinda Gates Foundation remains one of the most obvious organizations to look to, if only because it is one of the only organizations funding projects of this type.
"We're happy with our test … but if we could get a lot of money then we'd be able to say to TwistDx: 'Make something and sell it for us,' and they would be happy to do so, I think," Boyle said.
"How much money is out there, how much market is there?" Hashsham added. "We never have good numbers on those things. We have a good handle on what is being used, and there are many mutations on the human genetics side that are very easily doable, with no sample processing. But I think that by making something very low-cost, you are already saying that you have to sell a lot before you can make some money. That's the biggest challenge, I think. And if you are a smaller company, you are never going to reach to that larger market without partnering with someone."