Using PCR to diagnose a severe respiratory bug

Rhinoviruses and enteroviruses are common respiratory viruses, and generally just cause a bit of a snuffle. However, in winter 2014, an outbreak of enterovirus D68 led to around a thousand confirmed cases of severe illness and 14 deaths in the US. A team of researchers at Washington University School of Medicine in St. Louis have developed a diagnostic test based on the polymerase chain reaction that can detect the virus quickly. The research was published in the Journal of Clinical Microbiology.

Child sneezing

The team sequenced and published the genome sequence of the EV-D68 virus that was found in St Louis, Missouri. They used computer software to compare a large number of DNA sequences from all variants of D68 found in public databases with each other, and with sequences from other viruses. They selected a segment of the VP1 gene that is common to all the known D68 subtypes, but which isn’t seen in any other viruses, and used this to create a strain-specific RT-PCR assay. This was faster than existing assays, producing results in just a couple of hours rather than several days, and was more sensitive than commercially-available diagnostic tests.

“Commercial tests for respiratory viral infections typically don’t distinguish between rhinoviruses, which cause the common cold, and enteroviruses, and within each of those groups there are many different types,” says senior author Gregory A Storch.

The PCR-based test did not detect any other enteroviruses or rhinoviruses tested, and did not miss any of the variant strains of EV-D68, such as the first EV-D68 strain (Fermon) identified in California in 1962, even at low levels. However, any mutations of the virus in the future could restrict the effectiveness of the test.

“There are a range of D68 viruses, and our assay was designed to detect them all,” says Storch. “We received many samples of enterovirus from other hospitals and ran the test blindly on all of them. In the viruses we looked at, the test worked 100 percent of the time. It only detected EV-D68 strains, and it did detect all of them; the test didn’t detect any of the other enteroviruses or rhinoviruses.”

The test could be used as a template by other labs to create their own tests in the event of another outbreak, or could create more tests for other viruses whose genomes have been sequenced.

“Having a tool to identify which cases of respiratory illness are actually EV-D68 is an advantage for public health,” says Storch. “These kinds of tests help treatment decisions because it is important to know that the patient doesn’t have influenza or another disease that might require a specific treatment. It’s also important in a hospital for preventing infections because doctors take patients with one particular virus and keep them apart from patients infected with other infectious agents.”

Tags:

Add a comment

You must be logged in to post a comment.

No comments yet.