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Outbreak of EV-D68 Detected by Cross-reactivity in GenMark Respiratory Viral Panel

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NEW YORK (GenomeWeb) – Researchers at the University of Minnesota Medical School have used Sanger sequencing of pediatric patient samples to demonstrate that certain "low positive" results from a commonly-used PCR-based respiratory virus panel from GenMark Diagnostics were actually enterovirus D68, not human rhinovirus.

The results of the analysis were published online in PLoS One last week.

The 2014 outbreak of EV-D68, a non-polio enterovirus that belongs to the picornavirus family, is confirmed to have sickened 1,153 people in 49 states, and there were likely millions of mild EV-D68 infections that remained undetected during this outbreak, according to the US Centers for Disease Control and Prevention.

The CDC developed an improved EV-D68 PCR assay in response to the outbreak, as reported by GenomeWeb. Although the Minnesota State Health Department can run that test, Shane McAllister, a physician and researcher in pediatric infectious disease and molecular virology at UMN and co-author on the study, told GenomeWeb in an interview that sequencing the samples was a quicker option for his group.

"The portion of the virus that we sequenced is within the same region that the GenMark platform targets," McAllister explained. "We know that it is not the most specific area to distinguish between different viruses within the family [of] picornaviruses," he said.

Human rhinovirus is also in that viral family. "They all share a pretty close homology within the untranslated region, which is what we targeted, and what the GenMark platform targets," McAllister said.

GenMark noted that prior to the EV-D68 outbreak in 2014, the firm's analytical testing with a commercially available strain of EV-68 from ATCC did not indicate that there was any cross reactivity between its XT-8 respiratory viral panel (RVP) assay and that viral strain, the firm told GenomeWeb in an email.

During the outbreak, GenMark received reports of potential cross reactivity between HRV and EV-D68, a representative said. The company investigated the cross reactivity, confirmed that in many cases the HRV assay did react with the circulating EV-D68 strain, and subsequently updated its package insert.

Many of the samples in the UMN study came from children who had such serious respiratory symptoms that they needed to be admitted to the pediatric intensive care unit. These cases were also unusual because many of them were children who had no previous history of airway disease, such as asthma, or wheezing.

"We were seeing all of these patients coming in who were severely ill, who were coming up with rhinovirus on the GenMark platform, and that's just not consistent with what we know of most rhinovirus infections," McAllister said.

Of 49 samples in the study that were "low positive" for human rhinovirus using the RSV panel, 33 turned out to be EV-D68 after sequencing of the picornavirus 5' untranslated region.

The UMN group also identified 14 unique sequences in this sample set, and submitted these to GenBank.

It is possible the 2014 outbreak strain of EV-D68 may differ genetically from previous strains, which could in turn account for heightened virulence in children, the study noted.

Patricia Ferrieri, director of the infectious diseases diagnostic laboratory at UMN and a co-author on the study, explained in an email that the research suggests EVD-68 can evolve from standard reference strains and cross-react with human rhinovirus.

"I do not recommend using low positive results as surrogate markers during epidemics of EVD68," Ferrieri said. "However, it may be a tip off to pursue with more specific assays."

She also noted that in general the GenMark panel, which detects 14 respiratory viruses, is "quite sensitive and specific."

McAllister concurred. "I don't think that there is a flaw in the technology — in actual fact I have nothing bad to say about the GenMark platform, we continue to use it and find it very useful — but I think this is a good example of how clinicians need to very cautiously interpret lab results in light of the clinical picture that they see in the patient in front of them."

The case also provides an "opportunity to appreciate the limitations of the technology as it is now, and to improve it," McAllister said. "This may be telling us that what we need in future iterations of this technology is to be targeting sequences that are much more specific for different members of this large viral family."

GenMark has been developing a next-generation platform based on its eSensor detection technology. Called ePlex, this platform will also integrate automated extraction and amplification. It is expected to launch in Europe later this year and in the US in the first half of 2016, and the firm is designing a menu of seven panels.

"As we look to the future with our ePlex system, we are working closely with experts in respiratory testing to determine the most clinically meaningful way to address the detection of enteroviruses and rhinoviruses in respiratory specimens," the company told GenomeWeb.

GenMark reported a 51 percent increase in fourth quarter revenues last month. The firm placed 38 XT-8 analyzers in end-user laboratories in 2014, bringing the total installed base to 540 instruments in the US.

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