- New sensitive droplet digital PCR (polymerase chain reaction) assay
found to be accurate in detecting hepatitis B virus (HBV) infection by
measuring cccDNA in serum, single liver cells as well as processed tissue
- HBV infection is
associated with increased risk of progression to hepatocellular cancer
(HCC); recently several new treatments targeting cccDNA have been
established to eliminate HBV infection.
Latest droplet digital PCR (ddPCR) assay
improves detection of cccDNA and is highly sensitive compared to current
methods to detect HBV infection according to a recent study conducted at the
Center for Gene Diagnosis, Zhongnan Hospital of Wuhan University, Wuhan, China.
The findings of the study appear in The Journal of
Why Droplet Digital PCR
is Better Than Current Methods to Diagnose HBV Infection
- It is important to diagnose HBV
infection early due to the increased risk of progression to liver cancer.
Several new targeted treatments are now available to clear HBV infection,
thereby preventing or reducing liver cancer risk, making early diagnosis
all the more critical.
considered the gold standard to diagnose HBV infection, Southern blotting,
and other assays have certain limitations like less sensitivity than
ddPCR in detecting low levels of cccDNA.
"With droplet digital PCR assay, 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," explained lead investigator Song-Mei Liu, MD, PhD, of
the Center for Gene Diagnosis, Zhongnan Hospital of Wuhan University, Wuhan,
methods require obtaining a liver biopsy which is an invasive procedure
and carries risk of bleeding and infection. This assay can be done on serum
samples, which is not as invasive as liver
Additionally it is able to accurately detect trace molecules
such as viruses.
‘Estimating serum cccDNA is of great value in earlier diagnosis of HBV infection, prediction of risk of progression to HCC, targeted intervention, and assessing treatment efficacy.’
"Compared to liver biopsy, serum can be
obtained noninvasively, is widely used for clinical diagnostic purposes, and
has a homogenous cccDNA distribution. The assay also improves the limit of
detection of cccDNA," noted Dr. Liu.
Findings of Study
- Droplet Digital PCR
(ddPCR) was helpful in detecting which patients could be harboring
hepatocellular cancer. The team found that almost 90 percent of 68 HCC
patients were cccDNA-positive in comparison to 53 percent of 79 patients
without liver cancer.
serum cccDNA viral copy numbers i.e. viral load, was found to be more in
HCC patients compared to patients without liver cancer.
evaluation of serum cccDNA and HBV-DNA was able to distinguish liver
cancer patients from non-HCC patients. "This implicates cccDNA as a
risk factor for HCC," said Dr. Liu.
- The scientists
were also able to establish that serum cccDNA correlated positively with
levels of cccDNA measured in liver samples indicating measurements could
be made on serum samples.
"Serum cccDNA is indeed a much better and
useful diagnostic marker than intrahepatic cccDNA," said Dr. Liu.
Recently, several new antiviral strategies targeting cccDNA have been found to
improve HBV clearance.
Thus, the study suggests that
droplet digital PCR and HBV DNA estimation is superior to current methods in
detecting HBV infection earlier so that targeted treatment can be started
earlier and reduce liver
In an accompanying commentary Fan
Shen, MD, PhD, of the Department of Laboratory Medicine and Pathology,
University of Alberta, Edmonton, AB, Canada, observed, "Quantification of
serum cccDNA and HBV-DNA is an effective way to discriminate HCC patients from
non-HCC patients with a sensitivity of 74.5 percent and a specificity of 93.7
percent. This is an important finding and could lead to earlier diagnosis of
HCC and more accurate prediction of chronic HBV progress in a noninvasive manner."
As per WHO estimates, 257 million individuals
were infected with HBV globally, and in 2015 there were 887,000 deaths, mainly
due to liver complications. Chronic HBV infection can be managed with antiviral
medications such as tenofovir or entecavir
to clear infection.
Is Droplet Digital PCR Assay?
Digital PCR (dPCR) is a novel technique for
accurate quantification of nucleic acids. It employs similar assay reagents
that are used in standard analog measurements, but measures the total number of
individual target molecules such as viral DNA particles in a digital format,
making it useful in many applications that require high degree of sensitivity
and availability of samples is restricted.
In conclusion, digital PCR method could improve
diagnosis of early HBV infection so that early targeted treatment can be
administered to reduce or delay HCC risk with an improved outcome for patient.
- Fan Shen et al. "Hepatitis B Virus Covalently Closed Circular DNA-Selective Droplet Digital PCR", The Journal of Molecular Diagnostics (2018). DOI 10.1016j.jmoldx.2018.03.001