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Isothermal Assay from PATH, Hutch, TwistDx Rapidly Detects Multiple HIV-1 Subtypes


Scientists from Seattle's PATH institute, the Fred Hutchinson Cancer Research Center, and Alere subsidiary TwistDx have published a paper demonstrating the rapid detection of HIV-1 proviral DNA using assays based on TwistDx's recombinase polymerase amplification, an isothermal amplification method.

According to the researchers, their work is the first example of an isothermal assay that consistently detects all major HIV-1 global subtypes. Taking into account the qualities of the RPA technology that make it particularly suitable for point-of-care use, the researchers believe their assay could prove a minimally instrumented tool to improve diagnosis and treatment of HIV-1 in infants in the developing world.

"We're in a good place [in that] this assay could be used anywhere globally and be able to detect the HIV strains that are prevalent in that particular region," David Boyle, a senior research scientist at PATH and principal investigator for the effort, told PCR Insider recently.

PCR Insider first reported in 2011 that Boyle and colleagues from PATH and TwistDx were developing their assay under a grant from the National Institutes of Health (PCR Insider, 8/25/2011).

At that point, the researchers had developed an initial prototype of their test after looking into various isothermal amplification techniques and settling on the RPA method.

Several attributes of RPA make it attractive for use as an assay technology for decentralized testing in the developing world. The technique uses a recombinase to facilitate the insertion of oligonucleotide primers into their complement in a double-stranded DNA molecule — a process that can occur at a relatively low and constant temperature in the range of 25 to 42°C.

A strand-displacing DNA polymerase can then extend from the primer-bound complex to synthesize a new complementary DNA strand, and, as with PCR, the use of two opposing primers allows exponential amplification of the target sequence. The resultant amplicon can be detected either by monitoring fluorescence emission in real time with a fluorescence reader or simply by using lateral flow strips.

Since 2011, Boyle and his colleagues have worked to show that they could use RPA to design assays that could reliably detect a substantial number of strains of HIV-1, which is known for its extraordinary genetic diversity.

Specifically, as described in a paper published this week in the journal mBio, the scientists tested 63 HIV-1 specific primer and probe combinations and identified two RPA assays that target distinct regions of the HIV-1 genome: the long terminal repeat region, or LTR region, and the pol region.

"We took [HIV-1] subtypes A through D, and then looked at several recombinant forms, and found that the assays are highly tolerant to sequence variants," Boyle said. Indeed, the LTR primer set amplified 93 percent of 15 diverse viruses; and, in a larger screen, the pol primer set amplified 71 of 72, or 98.6 percent of variants tested.

"Both of these assays can detect down to three copies of proviral DNA using either [a fluorescence reader] for fluorescence detection or using a lateral flow strip for endpoint detection," Boyle said. The latter would likely be most ideal for decentralized testing in the developing world because it would eliminate the need for any instrumentation; however, numerous simple, portable, and low-cost fluorescence readers are commercially available or under development.

Another positive attribute of the RPA-based assays is that they can be completed in less than 20 minutes when performed at the optimal temperature range of 25 to 42°C. This is important because, as the researchers noted in their paper, when diagnostic tests administered in remote locations are sent to centralized laboratories in the developing world, a significant percentage of patients never return to the original testing site to obtain results or begin appropriate treatment, even if the turnaround time is a matter of days.

Boyle and colleagues have an ongoing development agreement with TwistDx and still have about a year of funding left on their NIH grant. During this time, the group plans to investigate ways to turn the basic assay into a commercial product.

"Now we're working on taking a variety of low-cost, simple extraction methods and seeing which one dovetails best with the RPA assay," Boyle said. "We'll identify the best sample collection and preparation format to fit into the assay, which we've now shown as having great performance."

A commercial assay kit would most likely need to work with blood collected from infant heel sticks. "We're thinking about … either direct lysis of whole blood, because the assay is fairly tolerant to whole blood … or enriching for the cell type that actually harbors the viral DNA. We may be able to get better performance that way. It comes down to what works best, and also the cost, and how easy it would be to take these different components and have them manufactured and made into a test."

Another pertinent issue is temperature control. Although RPA works in a temperature range that is a fairly common in much of the developing world, the assay may fail, or at least work much more slowly, if the ambient temperature nears the lower end of the range.

In many other molecular assays, such as PCR or even some isothermal methods, "the primers are designed to be optimal at a very specific temperature," Boyle said. "With RPA, because it's enzyme-mediated, we don't see that. If it's a lower temperature, it works, just more slowly. It might be that if we incubate an assay at 25 degrees [Celsius], but incubate it for 30 to 40 minutes, we see the same performance in terms of sensitivity and specificity, and we're investigating this."

But temperature fluctuations can also be a major problem in underdeveloped countries, and as such the scientists are also investigating non-instrumented methods for maintaining a constant incubation temperature.

"That's kind of the cornerstone of the rest of our work," Boyle said. "We've already identified some chemical heating sources … where if the temperature is below 34 degrees [Celsius] we can incubate the reaction independently of power, while keeping it in a range where it doesn't overcook."