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New Rapid RT-PCR Assay Detects All HIV-1 Groups


NEW YORK (GenomeWeb) – As part of a larger project aimed at producing a handheld molecular diagnostic for use in the developing world, researchers at Dartmouth have developed an assay to rapidly and reliably detect all subtypes of HIV-1 in plasma samples.

The molecular work was done in the lab of Gregory Tsongalis, a professor of pathology at Dartmouth-Hitchcock Medical Center and the Geisel School of Medicine at Dartmouth. Researchers there have developed a test that uses intercalating dye RT-PCR and melt curve-based controls to detect the most common HIV-1 M, as well as O, N, and P subtypes.

In an interview with PCR Insider, Tsongalis explained that assay development is one arm of a collaborative project with Axel Scherer, professor of nanotechnology and head of the global health initiative at the California Institute of Technology. Scherer was a co-founder of Helixis, a company acquired by Illumina in a deal worth up to $105 million in 2010, which is now part of the PCR assets Illumina is hoping to sell.

The team intends to port the HIV-1 assay onto a handheld device that they are developing in parallel. They will ultimately commercialize this device as well as the assays it will run. The intellectual property is currently held by Caltech, and Tsongalis implied the group intends to form a startup to bring the technology to market.

"There are instruments here in the US — the Cepheid gene expert, the Biofire, the BD max — all those things that are kind of turn key, but the requirements for electronics and power would really not cut it in the developing world," Tsongalis said. "We're trying to develop a very similar device but using limited power." A prototype instrument described in a recent review authored by the group runs on a 9-volt battery, he said.

The new HIV-1 assay also may be useful to the developed world as is, since there are not many inexpensive qualitative assays currently on the market. "Roche and Abbott [for example] have assays available for quantitative, but there really aren't any good qualitative assays available," Tsongalis said.

According to the description of the assay, published in Experimental and Molecular Pathology, it requires viral RNA extraction using Qiagen's EZ1 robotic system and employs the Bio-Rad iTaq Universal SYBR Green One-Step kit. The group also used the AcroMetrix HIV-1 panel from Life Technologies to establish the limit of detection for the assay.

Armored RNA from Asuragen was used as the extraction and RT-PCR control. Recently, this standard was licensed to Becton Dickinson for molecular diagnostics development.

"One of the issues when you're developing RNA-based assays is the type of control you use and how stable it is," Tsongalis said. "The armored RNA seems to be really good with respect to those characteristics. It's not cheap ... but for us right now it seemed to be the best way."

In validation studies, the assay showed 100 percent concordance with previously quantified patient plasma specimens, including 48 negative and 5 positive samples.

A step toward democratizing MDx

Clinical labs can now adapt the published qualitative HIV-1 assay. But, in its current incarnation, the requirement for power and complex equipment makes it less useful in low-resource settings.

Tsongalis and Scherer recently outlined such requirements for molecular diagnostics in an American Journal of Clinical Pathology review article titled "Democratizing molecular diagnostics for the developing world."

The review cites a series of papers on global diagnostics in Nature, published as a supplement in 2006. That collection describes the results of a two-year project to determine the potential impact of molecular diagnostics in the developing world.

In the ensuing eight years, many designs for diagnostic devices for low-resource settings have been described, but, as recently reviewed in Lab on a Chip , none has yet come to fruition in the sense of a product for sale or available to clinicians and patients.

Tsongalis said this is not for lack of ideas or capital.

In fact, researchers have needed to be incredibly creative to adapt molecular techniques to hot climates with unreliable electrical and water supplies, Tsongalis said.

"When we started doing this I said to the people in my lab ... 'for this project only, forget everything that I've ever taught you, forget everything that we've ever done in the lab, because it's not going to work in this project, you have to think completely outside of the box.'"

One issue, highlighted in the AJCP review, is that in some locations where researchers would hope to use these molecular diagnostic devices, the thermocycling start temperatures might be below ambient temperature. They must also make reagents lyophilizable, eliminate the need for cold storage, and increase the shelf life for high temperature and high humidity environments.

However, developments in the field of PCR have also impressed Tsongalis, and he is hopeful. "It's unbelievable the things that people are trying, and its even more unbelievable the things that are actually working. Before this project, I would have never even dreamed about trying some of these things," he said.

As for the timeline to a low-resource point-of-care molecular diagnostic, Tsongalis said perhaps part of the problem is that researchers have to work around or license existing intellectual property when developing new devices.

It also simply takes time, he suggested. "Starting from scratch with a project like this and trying to develop something, the ideas are great and things come together nicely, but it just takes a while to get the right pieces in place. Not just for the instrument, but also for the reagents for the different assays, the sample prep, what type of sample are you going to use, things like that. There are probably a thousand moving parts that all have to come together."

The global health incentive is tremendous. The consensus in 2006 was that 12.3 million disability-adjusted life years could be saved by the introduction of the appropriate diagnostics in the developing world. "It will happen. There are groups that are getting closer and closer, and it will happen," Tsongalis said.