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PATH Researchers Develop Nucleic Acid Assay Platform for Low-Resource Settings


By Molika Ashford

An electricity-free heater based on exothermic chemical reactions can successfully incubate an isothermal nucleic acid amplification assay with results equivalent to commercially available PCR instruments, researchers at nonprofit international health agency PATH have shown.

Their results, published in PLoS One last month, reflect the group's ongoing work to create an "equipment-free" nucleic acid assay for molecular diagnostics in resource-poor areas, which lack the power and instrumentation necessary for current nucleic acid tests.

According to the study authors, this lack is a critical barrier to effective treatment for many infectious diseases endemic to these low-resource settings, including malaria, HIV, tuberculosis, and influenza.

"Healthcare facilities in rural areas commonly have only basic equipment, and health workers have limited training and little ability to maintain equipment and handle reagents. Reliable electric power is a common infrastructure shortfall," Paul LaBarre, one of the study authors, told PCR Insider this week. "Despite multiple attempts to develop small, portable, low-cost, instrument-free [nucleic acid amplification techniques] that can be used in [low-resource settings], none are commercially available."

The researchers note that one of the primary barriers to the availability of NA assays in resource-poor areas is the complexity of PCR amplification. Significant advances in instrument-free isothermal amplification techniques open the possibility of simpler NA testing than with PCR-based assays. However, LaBarre noted, almost all nucleic acid amplification techniques, or NAATs, require electricity.

The electricity-free non-instrumented nucleic acid, or NINA, heater the group tested in its paper is a step toward its ultimate goal of creating a full NINA test platform that requires "no electrical power, no batteries, and no external reagents," the authors wrote.

Creating a full kit will require similar advances in eliminating the power requirements of sample preparation, reagent storage, and readout. "PATH has parallel projects and collaborations that are focusing on removing the requirements for electricity at other steps in the NAAT process – specifically sample preparation and detection," LaBarre said.

The group's first heating prototype, discussed in the report, used loop-mediated amplification as the model method, and Plasmodium falciparum, the pathogen that causes malaria, as a model diagnostic target, with heat provided by exothermic chemical reactions like those used in camping hand warmers and ready-to-eat meal systems.

Tested against two reference heaters, PerkinElmer's GeneAmp 9600 and Qiagen's ESE-Quant Tube Scanner, the NINA system incubated LAMP assays using dilutions of P. falciparum DNA with no discernable difference from the commercial PCR devices, the researchers reported. For the GeneAmp 9600, results were "virtually identical," according to the authors.

According to the report, the prototype uses an exothermic reaction of CaO and water, combined with an engineered phased change material — in this case a hydrogenated fat compound with a melting range centered on 65 °C — to help maintain a steady temperature in the heater. An insulated thermos provided the housing for the heater, with a bottom chamber holding the exothermic reaction and the upper chamber containing the EPCM and reaction wells.

Ten runs of the prototype showed minimal variation in temperature, and the heater reached the optimal incubation temperature for the test in 15 minutes, maintaining with "minimal drift" for 60 minutes, the study reported. The mean temperature drift over all runs was 2 °C.

According to the researchers, the temperature consistency appeared more uniform than what was shown in earlier tests of hand-warmer-based devices, with "far less variation than any similar devices."

The tests were performed successfully with commodity grades of quicklime, they reported, making the disposable materials for the test (CaO, water, and PCR tubes) very inexpensive. Though the EPCM used in the study is not as readily available as CaO, they wrote, similar materials have been used in US consumer products, so local, affordable production in the developing world "should be feasible."

The researchers also tested their CaO-based system against a conventional battery heater, to demonstrate cost savings. Their analysis showed that a CaO heater was several times less costly than mass-produced dry-cell batteries. According to LaBarre, the heater's CaO cartridge should be less than $0.50, and the group's overall cost target is approximately $1.50 for the kit to be competitive with rapid diagnostic tests.

Though the PATH team did not design its study to rigorously examine the malaria assay used in the heating tests, their results suggest that sensitive quantitative assays with high dynamic range are possible. LaBarre cautioned that the research is at an early stage, but said the study has demonstrated that non-instrumented, electricity-free amplification is "non-inferior" to heat-block amplification. "We are planning a more rigorous study to assess the NINA performance with regards to these things," he said.

To provide quantitative results using LAMP, the study authors wrote, improvements would be necessary to the NINA system to facilitate precise timing. Hypothetical solutions they highlight in the paper include adding a window to allow visual monitoring while the reaction vessels are inside the heater. A parallel heater could provide an elevated "stop" temperature for the reaction, they wrote, as well as a chemical stop, or even a boiling water bath.

Most of the components for a full NINA kit already exist, the authors reported in their paper, and the team is now working to combine them into a field-ready instrument.

The team also reported that they explored the possibility of using different EPCMs to create heaters with temperature profiles suitable for other isothermal amplifications like exponential amplification reaction (EXPAR), nicking enzyme amplification reaction (NEAR), and recombinase polymerase amplification (RPA).

"We are currently collaborating with [researchers at] other institutions, who are assessing our NINA technology with other isothermal methods," said LaBarre. "LAMP is a robust assay but there are other isothermal methods — some, such as [helicase-dependent amplification] and [nucleic acid sequence-based amplification] are commercialized and others are in R&D phases."

According to LaBarre, the NINA platform is designed to be sufficiently flexible to shift diagnostics in low-resource settings to the preferred NAATs. He said the platform can be adapted for other relevant diseases, such as HIV, influenza, and tuberculosis, and the PATH team is collaborating with other groups to assess NINA's performance with HIV and other pathogens.

Because the heater test was not subject to strict environmental control and was performed in an air-conditioned laboratory, it is likely the system "was not challenged in the same way as it would be at its intended point of use," the authors wrote.

The next step is field testing, according to LaBarre. "We’ve conducted some non-clinical field evaluations of the form factor and NINA concept in India, Zambia, and Kenya … and are currently incorporating the results of that work into the next design," he said. "The next big step is clinical validation … to establish operational performance as well as clinical use values for [limit of detection], sensitivity, specificity, et cetera."

LaBarre said that PATH collaborators that are looking at the NINA platform for HIV testing were able to assess the heater under more complex and rigorous conditions — with a non-instrumented sample prep method and fresh whole blood samples rather than DNA dilutions, as well as under more variable temperature conditions.

"That’s probably the most significant improvements we made," LaBarre said. "Up to that test, we didn't know how it would perform [under different temperature conditions] … and we are very pleased with the performance."

While PATH does not itself commercialize or distribute products, LaBarre said the team has applied for US and international patents and is establishing collaborations with commercialization partners to "ensure the R&D efforts achieve the impact for which they were intended."

Have topics you'd like to see covered in PCR Insider? Contact the editor at mashford [at] genomeweb [.] com.

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