Clinics and research in hepatology and gastroenterology · 2025
Droplet digital PCR technique is ultrasensitive for the quantification of covalently closed circular DNA in the blood of chronic HBV-infected patients.
СтатьяПоддержка исследования: вне правительства СШАНазвание и аннотация: Перевод в очереди
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Singh Ravinder, Ramakrishna Gayatri, Sharma Manoj Kumar, Kumar Rahul, Gupta Ekta, Rastogi Archana, Tanwar Pranay, Sarin Shiv Kumar, Trehanpati Nirupama
Аннотация
BACKGROUND: 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.
METHODS: 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 (HBsAglo < 2000 IU/ml, n=11) and Group II (HBsAghi > 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.
RESULTS: Non-digested 3.6^10⁶ 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, HBsAglo patients showed a median of 8.5 copies/µl (IQR 2.75-9.75 copies/µl), and HBsAghi gave a median of 11 copies/µl (IQR 4-16 copies/µl) but with no significant difference between groups (p=0.093). Further, HBsAglo patients with low cccDNA copy numbers showed significantly higher fibrosis grades than HBsAghi (p=0.036).
CONCLUSIONS: 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.
METHODS: 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 (HBsAglo < 2000 IU/ml, n=11) and Group II (HBsAghi > 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.
RESULTS: Non-digested 3.6^10⁶ 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, HBsAglo patients showed a median of 8.5 copies/µl (IQR 2.75-9.75 copies/µl), and HBsAghi gave a median of 11 copies/µl (IQR 4-16 copies/µl) but with no significant difference between groups (p=0.093). Further, HBsAglo patients with low cccDNA copy numbers showed significantly higher fibrosis grades than HBsAghi (p=0.036).
CONCLUSIONS: 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.