A single-cell digital PCR method tailored for quantification of HBV DNA positive cells in liver biopsy tissues

Accurate quantification of hepatitis B virus (HBV) DNA in hepatocytes is essential for understanding the virus's biology and improving treatment strategies. Existing methods lack the resolution to assess infection heterogeneity at the single-cell level, which is critical for evaluating treatment efficacy and guiding personalized therapy.

We developed a single-cell infection detection PCR (scID-PCR) method for quantifying intrahepatic HBV-positive cells with single-cell resolution. scID-PCR was validated using HepG2.2.15 and Hep3B2.1-7 cell models to assess sensitivity, specificity, and reproducibility. As proof of concept, scID-PCR was applied to liver biopsy samples from five chronic hepatitis B (CHB) patients and one non-HBV-related hepatocellular carcinoma (HCC) patient.

Validation demonstrated that scID-PCR was highly sensitive, specific, and reproducible. scID-PCR detected HBV-positive cells in all CHB patients, including those with undetectable serum HBV DNA levels after 48 weeks of antiviral therapy. No cccDNA-positive cells were identified in the HCC patient. The proportion of HBV-positive cells offering insights into infection status and identifying patients more likely to achieve a cure.

The scID-PCR platform offers a novel and powerful approach to quantify intrahepatic HBV-positive cells with single-cell resolution. This method complements traditional serological markers offering a more nuanced view of CHB infection dynamics and supporting personalized treatment strategies.