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Study Validates Lumora's Isothermal Amp-based C. Diff Assay Using Heat Elution Sample Prep

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Scientists from UK-based molecular diagnostics developer Lumora have published a pilot study demonstrating how a new heat elution-based sample prep method can be combined with isothermal amplification and Lumora's bioluminescent assay in real-time (BART) readout technology to quickly and easily detect Clostridium difficile from stool samples.

Specifically, Lumora's method — dubbed HE-LAMP-BART for heat-elution loop-mediated isothermal amplification BART — showed 95.5 percent sensitivity and 100 percent specificity compared against a gold standard of cytotoxigenic culture and silica-based robotic extraction followed by PCR.

In addition, HE-LAMP-BART demonstrated higher sensitivity and comparable specificity to a commercially available isothermal amplification-based assay, Meridian Bioscience's Illumigene test.

Lumora, which primarily employs an outlicensing business strategy, is hoping that the study results will attract potential partners interested in implementing some or all of its assay workflow as part of a commercial test that could be faster, less-complex, and cheaper than existing molecular methods, Hayden Jeffreys, commercial director at Lumora, told PCR Insider this week.

"This is a really [good] example of our [workflow-based] approach … where we've tried to simplify the workflow as much as possible," Jeffreys said. "We've applied our technology and all the things we've learned through … pathogen testing for the food industry … and applied the same principles of cost reduction and simplicity for non-specialist users to come up with a very credible and well-performing assay."

Lumora spun out of the Institute of Biotechnology at the University of Cambridge in 2002 to further develop the BART technique, which it couples with isothermal amplification techniques such as LAMP for various molecular testing applications that it then attempts to license to partner companies.

Lumora's earliest and perhaps most significant partnership was its 2011 collaboration and licensing agreement with 3M, which now uses BART in its food safety testing assays. But Lumora has also been attempting to break into the clinical diagnostics space, inking a partnership in April with the Foundation for Innovative New Diagnostics to develop a rapid, high-throughput malaria diagnostic combining BART with a LAMP-based malaria assay developed by FIND and Japan's Eiken Chemical.

In the meantime Lumora has continued to develop ancillary technologies to complement BART, and this year it filed an international patent for its heat elution sample prep process. This method, the company said, uses the power produced by heat lysis in a sealed vessel to drive elution through inhibitor-removing materials, obviating the need for complex and costly robotics or pumps and producing purified nucleic acids for downstream amplification-based assays.

This heat elution technology plays an integral role in the C. difficile assay workflow, which the company detailed in a paper published this week in PLOS One.

The scientists noted in this paper that although cytotoxicity assays for C. difficile are highly sensitive, they are quickly being replaced by molecular tests such as the Illumigene C. diff assay and Cepheid's Xpert C. difficile, both of which are cleared for marketing by the US Food and Drug Administration. However, the authors noted, the Illumigene assay, while fast and inexpensive, uses a seven-step, four-transfer process for stool-based testing; and the Cepheid assay, meantime, is highly sensitive and automated, but comparatively expensive.

The HE-LAMP-BART procedure described in the paper, however, uses a two-addition sample prep process like Cepheid's assay and can produce results in about the same amount of time — less than an hour — as both the Cepheid and Meridian tests.

Applying the heat elution method to a stool sample for C. difficile testing involves adding a fecal swab to a column containing a buffered cocktail of resins selected to bind fecal inhibitors of nucleic acid amplification. The column is then sealed, shaken, and placed into a second collection tube with a user first having broken off a bottom seal of the first column, allowing eluate to exit the column into the tube.

The column and the tube are then placed on a heating block at 100° C for 10 minutes, which lyses the bacterial cells and release the DNA into buffer and eventually into the second collection tube, leaving the amplification inhibitors behind. This purified DNA is then used to directly reconstitute lyophilized LAMP-BART reagents.

"It's literally sample in, DNA at the bottom to go onto the freeze-dried reagents," Jeffreys said. "As soon as [the tube] gets into the lab, all the lab tech has to do is break the seal of the bottom of the first tube and heat it. The interaction between the healthcare professional and stool sample is now only a swab — nurses and the lab techs love it, because anything you can do to reduce the interaction between you and a stool sample is going to make your life better."

Jeffreys added that once the sample is sealed and protected, it has about 48 hours of stability, enough time to move the sample to a centralized testing facility, if so desired. "We're looking to extend that to about 96 hours, and are forming some trials around that," he said.

In the PLOS One study, scientists from Lumora and Public Health England tested the HE-LAMP-BART method against several other well-known tests for C. difficile using 218 stool samples collected over a 17 month period and subsequently frozen. The positive or negative status of the samples had already been determined using a combination of several established methods including cytotoxic cell culture, yielding 111 positive results and 107 negative results.

Compared to the cytotoxic cell culture result, as well as to a Public Health England-developed multiplex PCR assay using silica-based robotic extraction, 106 of 111 positive samples were found to be positive using the HE-LAMP-BART method, resulting in five false negatives and 95.5 percent sensitivity. Meantime, the HE-LAMP-BART method identified all negative samples correctly.

The researchers also compared HE-LAMP-BART to the Illumigene test, cytotoxic culture, and the multiplex PCR assay on a subset of 75 challenging stool samples, so characterized by things like low copy number, solid stool, a high level of inhibitors, and the presence of blood.

The multiplex PCR assay had 100 percent sensitivity, but, as the authors noted, is a much more time-consuming and complex assay. Meantime, HE-LAMP-BART showed 76.6 percent sensitivity while Illumigene had 76.6 percent sensitivity. All tests showed 100 percent specificity in this experiment.

Finally, the researchers tested a further subset of 27 challenging stool samples using HE-LAMP-BART, Illumigene, Xpert C. diff, and an immunoassay-based lateral flow assay. Here, the Cepheid test showed the highest sensitivity with 96 percent, followed by HE-LAMP-BART at 92 percent, lateral flow at 76 percent, and Illumigene at 72 percent.

The scientists speculated that the Illumigene test, which has been reported as giving invalid results in blood-rich samples, suffered the same limitations in their experiments. Such particulates, they noted, do not interfere with the HE-LAMP-BART approach.

The authors noted that a full clinical trial is necessary to further determine the performance of the assay on stool samples from diagnosed C. difficile infections.

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