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Study Shows cccDNA Detection With Digital PCR Sensitively Detects Hepatitis B Virus


NEW YORK (GenomeWeb) – By using digital PCR targeting a unique viral DNA conformation, researchers in China have created a highly sensitive hepatis B virus assay in the hopes of providing early diagnosis and halting progression to chronic HBV and ensuing liver damage. 

The new method exploits the ability of digital PCR to detect a nuclear episomal form of the viral genome called covalently closed circular DNA, or cccDNA, in serum, single cells, and preserved tissue samples, as described in the Journal of Molecular Diagnostics earlier this month.

More than 2 billion people worldwide are infected with HBV with 378 million chronic carriers, and areas of Africa and Southeast Asia are disproportionately afflicted. Harboring the virus over time can lead to cirrhosis and liver cancer, and a study in 2015 estimated HBV to be among the top 20 causes of mortality globally, causing approximately 680,000 deaths per year.

The hepatitis B virus' life cycle includes formation of cccDNA, which serves as the template for transcription of HBV RNAs and gives rise to progeny viruses, Song-Mei Liu, the senior author on the JCM study and a researcher at the Center for Gene Diagnosis, Zhongnan Hospital of Wuhan University, Wuhan, China, said in an interview.

"Physical elimination of cccDNA in hepatocytes is regarded as a crucial step in developing a cure for HBV," Liu said. Conversely, the inability to eliminate cccDNA is also believed to be a critical reason why currently available HBV antiviral therapies can fail to achieve a functional cure.

HBV virions also contain partial double-stranded DNA, known as relaxed circular DNA (rcDNA), the structure of which is similar to cccDNA, Liu said. Distinguishing cccDNA from rcDNA is a challenge, and the low copy numbers of cccDNA – averaging 0.1 to 1 copies per hepatocyte in chronic HBV – create further obstacles for detection.

"The similar DNA conformation required us to design cccDNA-selective primers and specific probes, as well as Plasmid-safe DNase to selectively degrade DNA with free ends and to improve discriminatory power," Liu explained.

In the study, Liu and colleagues compared their ddPCR assay, which used the QX200 Droplet Digital PCR system and DG8 cartridges, with rolling circle PCR and Southern blot to evaluate the accuracy and sensitivity.

"Our method could provide an ultra sensitive serum cccDNA detection, and the limit of detection can reach single copy in a hepatocyte," Liu said. The method was also demonstrated on serum, single cells, and formalin-fixed, paraffin-embedded tumor issues.

Using the ddPCR-based method in clinical samples, the researchers further found almost 90 percent of the 68 hepatocellular carcinoma patient samples in the study were cccDNA-positive compared to 53 percent of 79 non-HCC samples. Serum of cancer patients also had more copies of cccDNA and were correlated with levels of cccDNA measured in liver samples.

But, while there are other technologies that use digital PCR for HBV detection, none use cccDNA.

Liu said there is a commercial test for cccDNA detection, the Invader HBV-DNA assay, which was originally developed by Third Wave Technologies, a firm acquired by Gen-Probe several years ago and is now owned by Hologic, but she asserted that serum cccDNA can not be detected with the assay below around 750,000 copies per milliliter.

On the other hand, cccDNA-selective ddPCR could detect cccDNA in a single hepatocyte cell as well as different clinical samples, and serum cccDNA could be quantified when HBV-DNA was above about 1,000 copies per milliliter, Liu said.

For now, the team is not working with any company or other industry partner, but it does have a patent on the method pending in China.

Liu and her colleagues will now continue to increase the sample size to confirm that serum cccDNA can distinguish hepatocellular cancer, set a threshold for diagnosis, and compare the results in patients with different HBV genotypes.

A commentary article in JCM noted that the ddPCR-based method of cccDNA detection could potentially have clinical applications, but the authors of the commentary said uptake of the method may be hindered by the throughput and cost of digital PCR systems.

Specifically, the authors wrote: "Although ddPCR is entering numerous laboratories in the world, the clinical translation is still awaiting [FDA] approval. This is, in part, because of the various drawbacks of ddPCR and the systems linked to the ddPCR machine. The current robot can only examine eight wells at once. The cost of the nonreusable cartridges is prohibitive."

In response, Lisa Jensen-Long, vice president of marketing at Bio-Rad's Digital Biology Group, pointed out in an email that customers who have created laboratory-developed tests for CLIA labs report the system to be very cost effective, "especially given the high cost of missed detection seen in other technologies, like qPCR."

The cost for the basic consumables and reagents to run ddPCR assays, including oils, Supermix, and DG8 cartridges, is around $3 to $5 per reaction, and the firm's QXDx200 with Auto DG provides the throughput of a 96-well format.

Bio-Rad's original eight-well manual droplet generator is adequate for much of the testing done in research, Jensen-Long said, while "the Auto DG is designed for the throughput and rigor required for IVD use."

Bio-Rad expects to submit its ddPCR system along with a chronic myeloid leukemia clinical test to detect BCR-ABL gene fusions in the next few months. The company has focused primarily on oncology applications for ddPCR but it sees opportunities in other diseases that service labs and third-party kit manufacturers are helping to advance through licensing ddPCR technology, Jensen-Long said.