Diagnostic Accuracy of Plate-Based Digital PCR Assays for Simultaneous Quantitation of Hepatitis B and D Viral Loads in Patients' Serum.

Objective: This study aims to evaluate the diagnostic accuracy of plate-based digital PCR assays that will accurately and sensitively measure HBV and HDV viral loads in patients' serum simultaneously.

Methods: First, we optimized the components and parameters for real-time PCR and RT-PCR assays to measure HBV and HDV levels. Next, we employed identical components and parameters in digital PCR and digital RT-PCR assays. Subsequently, we evaluated the accuracy and sensitivity of these assays compared to established techniques and assessed their efficacy by analyzing different serum samples.

Results: The technical sensitivity of the optimized digital PCR procedures was 1-5 copies/reaction for both HBV and HDV. Compared to real-time PCR and real-time RT-PCR assays, HBV and HDV quantitative digital PCR assays demonstrated strong correlation coefficients (R2HBV = 0.944 and R2HDV = 0.900). Substantial levels of agreement were indicated by Lin's Concordance Coefficient (CCCHBV = 0.963 and CCCHDV = 0.933). In addition, 2% (2/98) of the clinical samples had low HBV concentrations that the digital PCR could identify but not the real-time PCR. The HDV prevalence found in the study was 3.1% (3/98) by real-time PCR but 5.1% (5/98) by digital PCR.

Conclusion: In summary, the optimized digital PCR assays successfully measured hepatitis B and hepatitis D viruses in serum samples with low viral loads that real-time PCR assays could not detect.