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

objectiveThis 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.

methodsFirst, 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.

resultsThe 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.

conclusionIn 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.