Effectiveness of PCR primers for the detection of occult hepatitis B virus infection in Mexican patients

Abstract

BackgroundOccult hepatitis B infection (OBI) is defined as the presence of hepatitis B virus (HVB) DNA in the liver of HBsAg negative individuals with or without detectable viral DNA in serum. OBI is a diagnostic challenge as it is characterized by a very low viral load, intermittently detectable through time. Individuals with OBI can develop chronic hepatic disease, including liver cirrhosis and hepatocellular carcinoma. The aim of this work was to produce tools to improve OBI detection of the HVB genotypes prevalent in Mexico.MethodsWe designed and tested primers to detect OBI in serum samples by nested and real-time PCR. Conserved sites in the viral genome were determined by alignment of the most frequent HBV genotypes in Mexico (H, G/H, F and D) and primers spanning the entire viral genome were designed for first round and nested PCR. Primers were tested in serum samples of 45 patients not co-infected with hepatitis C virus or with HIV, out of a group of 116 HBsAg (-)/anti-HBc (+) individuals. Primers were also tested in a control group with chronic HBV. Nested PCR products obtained from HBsAg (-)/anti-HBc (+) were sequenced and used to design primers for real-time PCR (SYBR Green).ResultsThe most effective primer pairs to detect HBV products by nested PCR targeted ORF regions: PreS2/P, S/P, X/PreC, and C; while by real-time PCR they targeted ORF regions PreS2/P, S/P, X, and C. Out of the 45 HBsAg (-)/anti-HBc (+) patients tested, the viral genome was detected in 28 (62.2%) and 34 (75.5%), with nPCR and real-time PCR respectively.ConclusionPrimers designed for real-time PCR detected up to 75.5% of suspected OBI Mexican patients, with or without liver disease, which represents an improvement from previous PCR strategies.

Hepatitis B virus core–related antigen is useful for surveillance of hepatocellular carcinoma recurrence in a patient with occult hepatitis B virus infection: Case report

“…The association of OBI with hepatocarcinogenesis has been reported 4,10‐12 . In an analysis of 53 HBsAg‐negative patients with unexplained HCC, Wong et al reported that cccDNA was detected in liver tissue of 29 (47%) patients 13 .…”

Comparison of four extraction methods for the detection of hepatitis B virus DNA in dried blood spot samples

“…Core and S/pol regions were evaluated for detecting HBV DNA in qualitative PCR. These two HBV targets are frequently used in commercial assays to detect HBV in serum (Abbott RealTime HBV Assay and the Cobas TaqMan HBV test, version 2; Liu et al, 2019) and are employed for detecting occult HBV or acute cases (Liu et al, 2019; Sosa‐Jurado et al, 2018).…”

Hepatitis B virus core-related antigen is useful for surveillance of hepatocellular carcinoma recurrence in a patient with occult hepatitis B virus infection: Case report

“…The association of OBI with hepatocarcinogenesis has been reported. 3,[8][9][10] In an analysis of 53 HBsAgnegative patients with unexplained HCC, Wong et al reported that cccDNA was detected in liver tissue of 29 (47%) patients. 11 On the basis of these reports, it was presumed that our patient also likely developed carcinogenesis from OBI.…”