High-complexity labs can go a few different routes to bring on testing. They can run an already-approved protocol exactly as is, in which case labs do not need to seek EUA, the FDA suggested in webinars with labs.
However, the agency also seemed to suggest that any variations on the CDC protocol—even mixing and matching reagents from other kits, for example—would require labs to perform analytical and clinical validation and file for EUA. However, FDA has also referred labs to the agency’s frequently asked questions page, which was updated recently to include certain acceptable protocol modifications.
If labs choose to make their own tests, they can begin testing patients as soon as the assay is validated to the standards of FDA’s recent guidance and must then file for EUA within 15 days.
At this point in time, it appears that nearly every step in the overall workflow of the PCR (polymerase chain reaction) test—from sample collection and extraction to thermal cycling—has the potential to cause labs trouble.
Specifically, the clinical diagnostic workflow that needs to be validated entails reagents like inactivated virus or viral constructs, extraction kits and instruments, real-time PCR systems, as well as positive and negative control materials.
On a good day, reagents can be ordered, or are already on hand in the lab, but now, there are signs that demand may exceed supply during the current emergency.
Also, with a novel pathogen like COVID-19, just having the viral nucleic acids available to test if the assay is working has been the first challenge.
The FDA guides that labs should determine the limit of detection using 20 samples, and subsequent clinical evaluation should include 30 contrived clinical specimens. Getting viral material to do this first step has stymied some labs, while others have found workarounds.
For example, in Seattle and California—the U.S. areas initially hit hardest by the COVID-19 outbreak—validation in two high-complexity labs is complete and testing has already begun.
Alex Greninger is the assistant director of the University of Washington Medicine Clinical Virology Laboratory, a service lab which launched last year. He and his team have managed to overcome the limiting step of obtaining viral RNA.
“We used positive specimens, which we are awash in now,” Greninger said in an email.
Meanwhile, Ben Pinsky and his colleagues at Stanford University are also well on their way to EUA and are testing patient samples in California. Pinsky, who is the medical director of the clinical virology laboratory at Stanford Health Care and Stanford Children’s Health said that to validate its test, the lab used dilutions of a highly positive clinical specimen that was quantitated using single-stranded DNA as a calibrator.
“Once we had access to the positive clinical specimen, validation proceeded very rapidly,” Pinsky noted.
Other labs developing tests may not have the same access to positive samples, however. There were many queries about this issue on the recent FDA webinars, specifically about whether labs might use naked viral RNA, or plasmids, and they were guided to obtain inactivated virus.
Greninger explained that spiking viral RNA into patient sample matrix does not make a good validation material, especially for limit of detection calculations, because it is quickly broken down by ribonucleic acids. “There is a reason viruses are encapsulated. … Snot is full of RNAses,” he explained.
The CDC and FDA have been directing developers needing to obtain viral materials to BEI Resources, a reagent provider established by the National Institute of Allergy and Infectious Diseases, as previously reported.
BEI has said that it has live and inactivated virus in stock. Some end users have reported that there is a one-time step involving registration in order to receive shipments of virus, which may be a small bottleneck.
There are also intermediate suppliers that can obtain live virus from BEI and inactivate it.
Dwight Oliver, medical director of the molecular diagnostics laboratory at the University of Texas Southwestern’s pathology department, said that his team started the process of creating a lab-developed test protocol this week.
“We plan to validate using whole viral genome, and [we are] waiting for that resource to arrive,” Oliver said in an email.
He said he believes there are many labs currently waiting for the same viral genome or attenuated virus that his lab is also requesting. “I fear getting the viral RNA to spike into samples and determine our limit of detection will not be fast, but it is too early to tell,” he said.