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CDC Begins Using New, Faster PCR Test for Enterovirus 68

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NEW YORK (GenomeWeb) — As a rare strain of enterovirus continues to sicken children nationwide, the US Centers for Disease Control announced this week it has developed an enterovirus 68-specific PCR test for surveillance.

The current EV-D68 outbreak began in August. Since then, the CDC has processed 1,163 samples from across the country, about half of which tested positive for the virus, the agency said in a statement this week. The new test will enable processing of about 1,000 samples of backlog, dating to about mid-September, within the next 10 days. Thus, the agency is also trying to mitigate the possibility that a glut of positive test results could lead to a public perception of a spike in cases.

In an interview this week, Steve Oberste, chief of the polio and picornavirus laboratory branch at CDC, told PCR Insider that up until last week the agency was following a protocol described 10 years ago in the The Journal of Clinical Microbiology. That method uses semi-nested PCR followed by sequencing in order to determine virus type.

The CDC's new test, which was developed over the past few weeks, is specific for the VP1 gene of enterovirus 68, and is a straightforward TaqMan real-time RT-PCR test, Oberste said. The method is also scalable and easily transferred to most common PCR platforms, which should now allow state and CLIA labs to take on more of the surveillance responsibilities.

There are commercial enterovirus assays currently available, Oberste noted, but they only determine whether any member of the virus species is present and don't provide genotyping data.

"We're still running, at least in this lab, a pan-entero PCR analogous to some of the commercial tests," Oberste said, "So we'll know if there's another enterovirus there. But in terms of the current outbreak, we won't be typing those right away, [but we] will probably get back to those for surveillance purposes later. Right now the question is, is it EV-D68, or not?"

Using semi-nested PCR and sequencing for typing gives abundant information about the subtypes of virus present in any sample, as well as about related rhinoviruses, but is, unfortunately, "a fairly long process, and, because of all the hands-on steps, it's hard to scale up to large numbers of specimens," explained Oberste.

The previous method had only been adopted by the CDC and a few larger state labs, Oberste said, while "a real-time PCR can be rolled out virtually anywhere nowadays." In fact, the CDC lab intentionally designed the new method so it could be more easily used at the local level.

The test was developed on the ABI 7500, in part because the influenza test that the CDC distributes is validated and has gone through FDA 510(k) clearance on that platform, Oberste said.

"We know that virtually all states and most other big clinical labs should have that platform," he said. "We're also trying to use some of the same enzyme master mix kits that they're using for flu," making the EV-D68 protocol "easier to adopt."

The new assay will enable the CDC to test 180 specimens per day, up from 40 per day using the previous method. Oberste said the faster, simpler test could also reduce the agency's workload as more state labs increase their testing.

The sample prep step remains standard RNA or total nucleic acid extraction, Oberste said. The most common specimens have been nasopharygeal swabs, which make up the bulk of samples the CDC receives for this type of testing and thus were the focus of validation studies. But he noted the lab has run the generic assay on "nearly every kind of sample," so other types might work as well.

This may prove useful should doctors wish to test for EV-D68 in samples from sterile sites like cerebrospinal fluid or plasma.

The current outbreak of EV-D68 began in August and is believed to have infected 691 people in 46 states and the District of Columbia. It causes a respiratory illness in children that can occasionally become severe, and is thought to be responsible for two deaths so far. Prior to the current outbreak, there were several smaller clusters of EV-D68 in the US and elsewhere, Oberste noted.

Although EV-D68 is considered one of the non-polio enterovirus strains, it has also been detected in a subset of recent cases of polio-like flaccid paralysis in children, although a causal relationship has yet to be established.

In cases occurring in California, two out of five children with neurological symptoms tested positive for EV-D68, as recently reported at a meeting of the American Academy of Neurology. In a more recent cluster of nine cases in Colorado, CSF samples from the children were negative for EV-D68, but four out of eight NP swabs were positive for the strain.

"In those California cases, the only detections were in respiratory samples, so it makes it really hard to know whether it was really causing the paralysis or if it was just a coincidental finding in a non-sterile site," Oberste explained.

"That's a question that's still up in the air ... If you have a highly prevalent organism it's hard to determine causation in a non-sterile site, [and] so far as I know, there has yet to be a detection in a sterile site sample like cerebrospinal fluid or serum. We are certainly looking, we and others, to see if there is a link ... Enteroviruses in general are known to cause neurologic illness, so it's plausible," Oberste said.

The agency now hopes to fend off possible perceptions of a spike in case numbers by getting the word out about the new, faster test.

"Because we've received so many samples in such a short time, there's a little bit of a lag in getting them finished testing," Oberste said. "Now we'll be able to test our samples more quickly, and presumably some of the states can test their own and will not send to us at all ... So there may be an apparent increase now that it is easy to test. We're trying to communicate that ... just so that people know that it's not necessarily a real increase, it's just simply catching up in some ways, or now that it's easier to test, it's easier to find," he said.

The lab is currently working on publishing the method, as well as putting the basic protocol on the CDC website. "That way, states and clinical labs can adopt it as an LDT," Oberste said.

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