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Study Shows that ddPCR for Clinical CMV Monitoring has Potential but is Premature


NEW YORK (GenomeWeb) — Virologists at the University of Washington and the Fred Hutchinson Cancer Research Center recently reported that in a small, retrospective study droplet digital PCR appeared to have no added benefit over qPCR for clinical monitoring of human cytomegalovirus load in transplant patients due to low overall throughput.

However, the group also previously published work demonstrating that ddPCR is beneficial for certain other niche viral detection applications, and noted that it may eventually trump qPCR for viral load monitoring as workflow improvements are made to future iterations of the platform.

In a study published online last month in The Journal of Clinical Microbiology, the group compared Bio-Rad's QX100 Droplet Digital PCR platform to a qPCR assay. They ran serial dilutions of CMV standards and found ddPCR had increased precision over qPCR at high viral loads, and was equivalently sensitive.

However, in a retrospective longitudinal analysis of 19 clinical samples, the researchers found that, at clinically relevant viral loads, there was no evidence of improved precision using ddPCR. The group concluded that ddPCR's lower throughput makes switching from qPCR for CMV monitoring a bad investment for clinical labs at this time.

In an interview with PCR Insider, principal investigator on the study Keith Jerome said he expects digital PCR to improve in the future, and has given his feedback to commercial manufacturers including Bio-Rad, which partially supported the group's study. However, he said that at the moment it simply takes too much time to run the numbers of samples usually batch-processed by clinical labs for CMV viral load monitoring.

"The throughput is five to eight-fold better for qPCR over digital PCR right now," he said.

"We can do 96-well plates with qPCR, so nearly 100 samples at a time. Digital is much more sort of one-at-a-time, much more hands on, with more handling steps. But those are engineering problems that will be solved," he added. "We try to give feedback to the manufacturers to say here's what we would like to see in the next-generation instruments, and I think that they're probably listening to that."

The cost of ddPCR platforms and consumables is not trivial, but Jerome suggested that the main cost of switching for a clinical lab would be due to this extra hands-on time, requiring expert personnel.

"I shudder to think about how many instruments we would need to have and how many people we would need to hire to do [CMV monitoring with ddPCR]," he said.

Jerome, a professor and head of the virology division in the department of laboratory medicine at UW, as well as a member of the vaccine and infectious disease division at the Hutch, was hopeful that ddPCR will improve in the future.

"The fundamental reality … is that digital PCR is more complex to perform than qPCR. That's not a result of the fact that the technology is inherently more difficult, and it doesn't necessarily need to be true forever … but the qPCR systems that we have now have been optimized over 15 or more years for laboratories like mine to use, whereas the digital systems are sort of first-generation platforms," Jerome said.

"All that said, if we could demonstrate that there was some advantage to our patients of doing this by digital PCR, we might say 'Well, it's worth it.' We treat the patients, we get them on the antivirals that they need sooner, or we don't mistakenly put patients on antivirals. ... But in the group of 19 that we looked at, we weren't able to show that there would be a clear advantage in patient management by digital PCR," Jerome said.

"We worded that kind of carefully," he added, "in that it doesn't necessarily say that there wouldn't be [an advantage with a larger sample], but it is clearly not such a huge difference that you can see it in a relatively small sample size of 19 patients."

CMV is a very common herpes virus that often causes no symptoms on initial infection, and then lies dormant in the host. "Most of us have been infected with it at some point in our lives," Jerome said. However, CMV can reactivate, and in situations of immune suppression, such as stem cell or solid organ transplants, viral reactivation can cause severe illness and death.

"It's very important to detect CMV early when it reactivates because we have effective antivirals, but they're much more effective if they're started before the patient actually has symptoms," Jerome said. "With our PCR test, we can detect the virus beginning to reactivate before the person is sick, and that allows the most effective therapy."

Jerome's study also showed that the ddPCR assay has improved intrassay and interassay precision compared to qPCR, particularly at very high viral loads, and that ddPCR was not susceptible to cross-contamination. In their study the researchers also optimized DNA extraction and used concentrated mastermix to show that ddPCR could be adapted to a clinical setting.

However, CMV monitoring can be a weekly requirement in transplant patients. Clinical decisions about initiating costly and toxic antiviral treatment need to be made at CMV plasma concentrations below 1,000 copies per milliliter, according to the study.

Jerome's group recently published an assay for chromosomally integrated human herpesvirus 6 in Clinical Chemistry, a method also developed on the Bio-Rad platform. That assay uses two-color detection to determine the ratio of HHV6 to a cellular gene, and thus identify situations in which the virus is integrated into the germline, Jerome said. In the case of HHV6, "[ddPCR has] really has allowed us to perform testing for a condition where there really hasn't been a diagnostic until very recently. It's a bit more of a niche right now than CMV is, but I think it illustrates the point that under the right set of circumstances a technology that is even a little more cumbersome can be incredibly useful."

Jerome's group also published last year demonstrating that digital PCR is very resistant to inhibition. He said that his group has just completed a study of stool — a sample type notoriously prone to PCR inhibition — to try to identify whether there are viruses in the stool virome that have been missed in the past. He said his group does a lot of gene therapy work as well, and "digital PCR is a fantastic way to look at the ability of our gene therapy approaches to actually knock down viral replication, because it is so precise."

Jerome previously published a review of viral diagnostics "in the era of ddPCR," which concluded digital PCR could be particularly useful for absolute quantification of target DNA sequences and rare mutant allele detection, applications that vendors have been heavily promoting. This review described the two major droplet platforms (Bio-Rad and Raindance Technologies), as well as the two plate-based platforms (Fluidigm and Life Technologies).

Although the recent Analytical Chemistry study only examined Bio-Rad digital PCR, Jerome said his group has also collaborated with Raindance and has found the results between the two droplet-based platforms to have "remarkable concordance, much better than you'd see between any two qPCR assays."

"It's very interesting, because I think it says that in the future digital PCR may be a major solution to the problem of variability between laboratories," he added. "That would be a tremendous contribution to the field and to the management of our patients."

Jerome acknowledged that researchers and clinical labs are only just beginning to find useful applications for droplet digital PCR. "CMV may not be one, at least right now, but I think digital PCR is here to stay and it's going to be a major contributor to healthcare in the near future," he added.