Droplet Digital PCR: A Powerful Tool for Accurate Quantification of Hepatitis D Virus RNA Levels and Verification of Detection Limits.

Reliable quantification of hepatitis D virus (HDV) RNA levels is necessary for initiating and guiding antiviral treatment. The aim of this work is to develop and validate a digital PCR method for the accurate quantification of HDV RNA, including evaluation of its clinical accuracy, especially for low-concentrated clinical samples. The reverse transcription digital PCR (RT-dPCR) development followed the standard procedure, including primer design, determination of linearity, calculation of recovery and the intermediate precision of the RNA extraction kits, determination of the limit of detection (LOD) and quantification (LOQ), droplet size measurements, conversion factor, and uncertainty budget. The World Health Organisation (WHO)-HDV international standard was used for RT-dPCR development. Commutability of the new method was explored, comparing RT-dPCR with quantification assays applied in clinical routine using clinical plasma samples covering a range of HDV RNA concentrations. The conversion factor from copies/mL to IU/mL was 0.77. LOD and LOQ of the RT-dPCR were 0.7 copies/mL (0.56 IU/mL) and 10 copies/mL (8 IU/mL), respectively. When evaluating the qualitative results of the clinical HDV samples at low concentrations, 31% of the HDV clinical samples tested negative by RT-qPCR were tested positive by RT-dPCR. The RT-qPCR and RT-dPCR quantitative data showed a good correlation with a standard deviation of ±1.12 log IU/mL. RT-dPCR is an accurate method for HDV RNA quantification that may serve as a complement to RT-qPCR, especially when accurate detection is essential for decision making in clinical settings.