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Abbott Lyme Disease Assay, Sample-Prep Method for Plex-ID Underscores Platform's MDx Potential

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NEWPORT BEACH, Calif. – Scientists at Abbott subsidiary Ibis Biosciences have developed an assay to detect and genotype Borrelia burgdorferi, the bacterium responsible for causing Lyme disease in humans, using Abbott's Plex-ID microbial identification system.

The assay, which is currently for research use only, has sensitivity and specificity comparable to that of the current gold standard diagnostic method, a two-tiered serology-based test that can take as long as a month to provide a definitive result.

As such, the new assay, which can be completed in several hours, may eventually enable diagnosis and treatment of Lyme disease before it moves to other parts of the body and causes more serious complications.

In addition, the assay is one of many with molecular diagnostic potential that Abbott is currently developing for its Plex-ID system, which recently received CE IVD marking and is currently pending marketing approval from the US Food and Drug Administration.

Mark Eshoo, director of new technology development at Abbott, discussed the new Lyme disease assay, as well as the detection and genotyping capabilities of the Plex-ID system, in a presentation yesterday at Cambridge Healthtech Institute's "Innovative Sample Prep & Target Enrichment in Clinical Diagnostics" meeting, held here this week.

In his talk, Eshoo explained that there are currently no highly reliable molecular diagnostic tests for Lyme disease. It is the most common vector-borne disease in the US and can result in a chronic infection that triggers myriad other health problems in a patient.

Lyme disease patients may present with relatively nebulous, flu-like symptoms, making diagnosis difficult in the absence of the characteristic erythema migrans "bull's eye" rash – which doesn't always appear, or may be atypical – underscoring the need for a reliable molecular test for B. burgdorferi, Eshoo said.

Abbott has been developing such a test for its Plex-ID platform, which has been shown to be able to identify a broad range of microorganisms directly from clinical samples with a high degree of sensitivity and specificity.

The platform is based on technology that Abbott originally brought on board when it acquired Ibis Biosciences in 2009. In general, the system combines automated sample preparation, broad PCR amplification, and electrospray ionization mass spectrometry of DNA amplicons to identify base composition based on molecular weight.

The goal of the broad-range PCR – which stands in contrast to the current trend of making PCR assays as specific as possible – is "to just make an amplicon … using unbiased PCR primers," Eshoo said. Then, the ESI-MS can differentiate amplicons with a high degree of specificity.

"It's not quite like sequencing, but it almost has the same level of sensitivity," Eshoo said. "We don't know the order of the base pairs present, but we know how many of each we have."

In order to maximize the utility of the Plex-ID for identifying and genotyping B. burgdorferi strains, Abbott researchers developed a sample preparation method that would allow the assay to detect any strain of the bacterium that might be present.

More specifically, the sample prep protocol involves a post-DNA-extraction target-enrichment step using eight primer pairs to identify B. burgdorferi to the species level. The method is similar to nested PCR, but uses an isothermal amplification method with up to 50 primers flanking each target loci, 25 on each side, with six to 10 base pairs between each primer.

This approach "statistically improves our chances of detection, and may allow us to get the genotype," Eshoo said. "The idea is not to [exclude] anything [like off-target loci], but to make more of the DNA we want from [B. burgdorferi]."

The specific workflow for the Plex-ID-based B. burgdorferi assay involves collecting blood from a suspected Lyme disease patient, extracting nucleic acid, adding isothermal amplification reagents and incubating at the appropriate temperature, conducting PCR amplification, and finally running through the Plex-ID system for ESI-MS analysis.

The upshot of the sample prep method is that the assay can detect the target loci even in a high background of human DNA by selectively amplifying B. burgdorferi markers.

Eshoo said during his presentation that running the assay with the specially designed sample prep method improved its detection capabilities compared with a traditional sample prep protocol. He also told PCR Insider in a follow-up interview that the approach could be applied to other molecular diagnostic methods. Although he didn't elaborate, it's easy to envision how such a protocol could be used in front of another highly sensitive and specific molecular analysis method: next-generation sequencing.

As for the performance of the Lyme disease assay, Eshoo and other Abbott scientists collaborated with researchers from Johns Hopkins University to evaluate its performance on clinical samples that had been diagnosed using the classic serotyping method.

Eshoo provided a snippet of early data, but declined to provide more detailed metrics due to the fact that results from the study are scheduled to be published in a peer-reviewed journal within weeks. However, he noted that the assay compared favorably with the gold standard, and was able to detect as little as one genome copy of B. burgdorferi per milliliter of blood, prior to seroconversion, with "minimal extra steps" to the typical workflow.

Further, using the assay, the researchers have thus far been able to genotype nearly 80 different strains of the bacterium from samples obtained from ticks, the vector for B. burgdorferi.

"The value of genotyping for B. burgdorferi is still in question." Eshoo said, but added that this work demonstrates the power of the Plex-ID platform to specifically identify microorganisms down to the sub-species level from a single sample.

"Even if there is a mixture of genotypes in one sample, this platform can identify that," he said.

The genotyping capability of Plex-ID was further demonstrated in a study published last week in PLoS One, in which researchers from Abbott, Case Western Reserve University, and other institutions demonstrated how PCR/ESI-MS on the Plex-ID, coupled with repetitive-sequence-based PCR, could genotype multiple strains and help characterize regional molecular epidemiology of Acinetobacter baumannii, a burgeoning hospital-acquired infectious agent.

Eshoo told conference attendees that the Lyme disease assay is for research use only, and that "there is some talk" at Abbott about commercialization. However, he noted that the company "has a lot on its plate right now … attempting to get [Plex-ID] approved by the FDA."

The company initially began marketing Plex-ID for use in applied markets, such as biodefense and forensics, and has struck multiple partnerships in those areas (PCR Insider 4/5/2010 and 2/23/2012).

However, Abbott has recently made headway in pushing Plex-ID into the clinical market, highlighted by the announcement in March that it is working with Dallas-based Genetics Laboratory to develop a Plex-ID test to rapidly detect microorganisms that cause orthopedic infections (PCR Insider, 3/8/2012).

In addition, earlier this month Abbott said that Plex-ID received CE marking in the EU along with three assays: Plex-ID Viral IC Spectrum, Plex-ID BAC Spectrum BC, and Plex-ID Flu (PCR Insider, 4/5/2012).

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