Development of an over-gap droplet digital PCR assay and evaluation of serum HBcrAg/qAnti-HBc as noninvasive markers for intrahepatic replication-competent HBV DNA in chronic hepatitis B patients with HBsAg loss.

This study assessed serum hepatitis B core-related antigen (HBcrAg) and antibodies to hepatitis B core antigen (qAnti-HBc) as noninvasive biomarkers for detecting intrahepatic replication-competent HBV DNA (repDNA) in chronic hepatitis B (CHB) patients who had lost HBsAg. The goal was to identify alternatives to liver biopsy for diagnosing residual HBV activity after HBsAg loss. A novel over-gap droplet digital PCR (ddPCR) assay was developed for sensitive intrahepatic repDNA detection. Over-gap ddPCR detected repDNA in 88.24% (135/153) of patients, which was a significantly greater percentage than traditional nested PCR (75.16%, 115/153; P < 0.01). HBcrAg was positive in 41.83% (64/153) of patients, and repDNA positivity was markedly greater in HBcrAg-positive patients (93.75%, 60/64) than in HBcrAg-negative patients (84.27%, 75/89; P < 0.01). In HBsAg-negative CHB patients, the HBcrAg level was moderately positively correlated with the intrahepatic repDNA level (r = 0.470, P < 0.001), whereas this correlation was not detected in liver cirrhosis patients. For predicting the intrahepatic repDNA status in HBsAg-negative CHB patients, the area under the receiver operating characteristic curve (AUC) was 0.773 for HBcrAg alone and 0.803 for qAnti-HBc alone. Notably, the combination of HBcrAg and qAnti-HBc significantly improved diagnostic accuracy, achieving an AUC of 0.913. The novel over-gap ddPCR assay exhibits superior sensitivity for intrahepatic repDNA detection. Serum HBcrAg, particularly when combined with qAnti-HBc, effectively predicts the intrahepatic repDNA status in HBsAg-negative CHB patients, providing a valuable non-invasive tool to replace liver biopsy for assessing residual HBV replication.