Highly Sensitive Detection of Hepatitis B Using ddPCR

Developing new, more sensitive assays to detect chronic infections, like those from the hepatitis B virus (HBV), is imperative to when trying to manage associated diseases such as cirrhosis and hepatocellular carcinoma (HCC).

Now, a new study from investigators at the Center for Gene Diagnosis in the Zhongnan Hospital of Wuhan University, China, describes a new HBV assay that offers advantages over currently used methods because it can detect closed circular DNA (cccDNA) in serum, single cells, and preserved tissue samples.

Findings from the new study were published in The Journal of Molecular Diagnostics, in an article entitled “A Highly Sensitive and Robust Method for Hepatitis B Virus Covalently Closed Circular DNA Detection in Single Cells and Serum.”

“The development of HCC is strongly associated with HBV. Recently, several new antiviral strategies targeting cccDNA have been established to improve HBV clearance,” explained lead study investigator Song-Mei Liu, M.D., Ph.D., chair and vice professor at the Center for Gene Diagnosis. “It is of great clinical significance to provide an accurate and sensitive approach for cccDNA detection. With this method, more and more patients with chronic HBV will have precision treatment available to prevent or delay HCC occurrence, and HCC in patients could be diagnosed at an earlier stage.”

According to the World Health Organization, 257 million individuals worldwide are infected with HBV, and in 2015 HBV resulted in 887,000 deaths, primarily due to liver complications. Chronic HBV infection can be treated with medications such as tenofovir or entecavir. In 2015, only 9% of individuals living with HBV infection were aware they were infected.

The new assay utilizes droplet digital PCR (ddPCR), which is highly sensitive and accurate for detecting trace molecules such as viruses. Using the ddPCR method, the researchers were able to detect cccDNA in serum, single cells, and formalin-fixed, paraffin-embedded tumor issues. Southern blotting, currently considered the gold standard for this purpose, and other assays have important limitations such as decreased sensitivity compared to ddPCR in detecting low copy numbers of cccDNA. Moreover, traditional methods often require invasive techniques like obtaining liver tissue through biopsy.

“Compared to liver biopsy, serum can be obtained noninvasively, is widely used for clinical diagnostic purposes, and has a homogeneous cccDNA distribution,” Dr. Liu noted. “The assay also improves the limit of detection for cccDNA.”

The new ddPCR method was useful in identifying patients that might be harboring HCC. The research team found that almost 90% of 68 HCC patients were cccDNA-positive compared to 53% of 79 non-HCC patients. Serum cccDNA copy number was found to be higher in HCC patients compared to non-HCC patients. Combined analysis of serum cccDNA and HBV-DNA distinguished HCC patients from non-HCC patients. “This implicates cccDNA as a risk factor for HCC,” Dr. Liu remarked.

Additionally, the scientists were able to confirm that serum cccDNA was positively correlated with levels of cccDNA measured in liver samples. “Serum cccDNA is indeed a much better and useful diagnostic marker than intrahepatic cccDNA,” Dr. Liu concluded.

Recently, several new antiviral strategies targeting cccDNA have been found to improve HBV clearance. Therefore, providing an accurate and sensitive approach for cccDNA detection is of great clinical significance for earlier diagnosis and HCC prediction, targeted treatment, and evaluating treatment efficacy.