Investigating SARS-CoV-2 variants of concern using single-well multiplex qPCR

Clinical pathology labs face a daily challenge of investigating positive COVID-19 samples to identify circulating variants of concern and assist public health monitoring efforts.

In this on-demand SelectScience® webinar, Dr. Ron M. Kagan, of Quest Diagnostics, Dr. Charles Cartwright and Dr. Lee Martin Smith, from SpeeDx, discuss the high-volume pathology laboratory SARS-CoV-2 variant testing experience and share how universal substrate-based technology (PlexPCR) can be applied to the evolving testing challenges of SARS-CoV-2, providing streamlined development of high-throughput solutions that can be readily multiplexed.

Read on for highlights from the live Q&A session or register to watch the webinarat a time that suits you.

Q: How important do you feel it is to have SARS-CoV-2 mutation information available within 24 hours of diagnosis?

RK: The actual SARS-CoV-2 mutations are not necessarily needed in treatment today. The mutation detection is more of a surveillance tool or perhaps used to distinguish reinfection from persistent infection. I don't see a need to have it available within 24 hours of diagnosis.

Q: Do you advise gene sequencing for every patient or only randomly?

RK: Gene sequencing is a surveillance tool. I think it's estimated that in the U.S. right now, we're sequencing somewhere between 5% and 10% of the cases. It would be tremendously expensive and not practical to sequence every patient, plus you need a certain minimum cycle threshold (Ct) to be able to sequence. As a surveillance tool, I think you need a reasonably random sampling across the surveillance area to be able to use it, but I don't think it's useful or practical to do every patient particularly because the results aren't currently guiding treatment.

Q: What's the limit of detection of your multiplex assays for Alpha, Beta, Gamma, and Delta variants? And can your E484K assay discriminate between Beta and Delta templates?

RK: E484K assay will detect any strain that carries E484K, which includes more than one of the variants of concern. Other variants such as the Brazilian P.2 variant and several other variants support the limit of detection. I don't have those numbers for the limit of detection in front of me, but I think it's in the order of the low thousands of copies per ml.

Q: Do you think, for this situation, the RNA-dependent RNA polymerase (RdRp) is still perhaps the most important for our assay target?

RK: It's a useful control in the assay. We need to have an internal control, a way to detect that the virus is present. Other targets could have been used. The design for the SpeeDx assay included RdRp. We're also looking at the Delta CQ between RdRp and the probes for the mutations to determine whether or not the signal for the mutation is relevant or not.

Q: Can you tell us if your assay will be FDA or EUA-approved?

CC: The intent here is to have the assay available as a research-use-only (RUO) product. In fact, I think we're launching that very soon. Then we will add the Delta, the second well assay to that I think within the next month or so.

There really isn't a legitimate pathway right now to have these assays cleared. The FDA is still working through an immense backlog. Our intent here is really to have people do essentially what the folks at Quest have done which is they buy the component, and then they essentially validate their own laboratory-developed test using our RUO components. That also gives us the opportunity to be much more flexible in adding additional assays to the pipeline because we don't have to go through all the regulatory hoops in order to make those available, and given that this is a moving target, that's the approach we decided to take.

Q: Are there any assays to detect a potential vaccine-resistant variant of SARS-CoV-2?

CC: What we know right now is relatively limited about the impact of these novel variants that are circulating, in terms of overall response or the success of vaccines. I think the laboratory data that's been generated has shown that the mutations present in a couple of these variants certainly at least diminish the ability of an infected individual to produce a neutralizing antibody of the same type that we would see in a wild-type strain. The question, of course, is that since protection against the virus is not solely determined by neutralizing antibodies but also by our cell-mediated immunity, and there is only limited data on that, I think it's very difficult to know whether any of these substantively impact vaccine efficacy.

Again, the purpose of the tests is to identify variants of concern and there isn't right now a variant that defines the vaccine. There's not a true immune escape variant. Continued surveillance is essential in case one of those emerges. But right now, the scope is essentially variants of concern. The primary difference between these appears to be the efficiency with which they can infect and the efficiency with which they can be transmitted to nonimmune individuals.

Q: How often is the variant panel updated?

CC: That depends on what the virus does as we progress through the continued pandemic and then whatever comes post-pandemic with COVID-19. Now, what seems to be happening is a convergence in these variants, where there are a limited number of amino acids within the spike protein that appear to be pivotal in either increasing transmissibility or potentially in partial immune escape. I think unless we see dramatic evolution of the virus with different amino acid substitutions, we will continue to refine our offering so that it enables rapid identification of all of the variants of concern by focusing on this limited number of amino acid changes.

Again, this all depends on the virus not necessarily on us or any other company. I think what we can say is that we have a commitment now to continue to monitor this and to put our R&D teams into action as soon as something is seen that potentially is going to develop into something that our assays don't currently identify. We will target that, and we will produce add-ons to the current product pipeline.

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