Droplet digital PCR technique is ultrasensitive for the quantification
of covalently closed circular DNA (cccDNA) in the blood of chronic
HBV-infected patients
Abstract
Covalently closed circular DNA (cccDNA) is a stable, episomal form of
HBV DNA. cccDNA is a true marker for the intrahepatic events in
controlled CHB infection. Quantifying cccDNA is critical for monitoring
disease progression and efficacy of anti-viral therapies. To standardize
the method, total HBV DNA was isolated from HepAD38 cells and digested
with three exonuclease enzymes to remove linear and relaxed circular HBV
DNA. Purified cccDNA quantification used ddPCR with specific primers.
Treatment-naive chronic hepatitis B virus patients (nCHBV, n=36) with
detectable HBV DNA and HBsAg, were grouped by HBsAg levels: Group I
(HBsAg lo < 2000 IU/ml, n=11) and Group II
(HBsAg hi > 2000 IU/ml, n=25). cccDNA,
HBV DNA and HBsAg, were quantified in plasma and compared between
groups. Correlation with clinical/histopathological features was done.
Non-digested 3.6 ^10 6 tet
-ve HepAD38 cells showed 316 copies/µl of total viral
DNA. After digesting the linear, integrated, and relaxed circular DNA
with triple enzymes, 15 copies/µl of cccDNA were detected. Similarly,
after DNA digestion, HBsAg lo patients showed a median
of 8.5 copies/µl (IQR 2.75-9.75 copies/µl), and HBsAg
hi gave a median of 11 copies/µl (IQR 4-16 copies/µl)
but with no significant difference between groups (p=0.093). Further,
HBsAg lo patients with low cccDNA copy numbers showed
significantly higher fibrosis grades than HBsAg hi
(p=0.036). We conclude that employing a combined approach utilizing
three exonucleases, cccDNA-specific primers, and ddPCR enables the
detection of cccDNA copies even in patients exhibiting low levels of
HBsAg and HBV DNA. This integrated method offers additional validation
as a surrogate diagnostic tool.