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.

Background Respiratory viral infections are an important cause of acute respiratory tract infections. They are caused by both Influenza and non influenza viruses. Respiratory viral infections are known to be associated with severe clinical outcome especially in the critically ill. A constant surveillance is needed for early etiological identification which can help in timely and appropriate management. Methods In this retrospective study, clinical records of all adult liver disease patients with clinically confirmed ARI, whose request for respiratory viral testing were received in the virology laboratory during September 2016 - March 2019 were reviewed. Respiratory viruses were identified by real time PCR on FilmArray 2.0 instrument (BioFire Diagnostics, Utah, USA) using Respiratory panel as per the manufacturer’s instructions. Results Of the 603 patients of liver disease with clinically confirmed influenza like illness, over all incidence of respiratory viral infection was 24.3% (n= 147). Infections by non-influenza viruses (87, 59.1%) were more than influenza group of viruses. Mortality was higher in non influenza group (43, 49.4%) as compared to influenza (24, 40%) [p=0.015] being maximum in Rhinovirus, 22 (32.8%). Two peaks were observed in both influenza and non influenza groups, first in the months of January and February and the other one in August and October. Conclusion With the emergence of SARS- CoV-2 it has now become imperative for a constant surveillance of the non influenza viruses for early etiological identification of the respiratory viral infection for proper and timely management in the critically ill.