Clinical establishment of a Laboratory Developed quantitative HDV PCR assay on the cobas 6800 high-throughput system

Question Hepatitis Delta Virus (HDV) is a negative strand circular RNA virus with strong self-base-pairing and high diversity between the 8 known genotypes (GT). Both of these features pose a considerable challenge for diagnostic workflows and many available PCR assays are characterized by considerable run to run and inter-laboratory variability. The aim of the study was to establish a quantitative real-time PCR assay on the open channel of a fully automated PCR platform (cobas6800, Roche) offering improved consistency and reliability.

Methods A primer/probe-set targeting a highly conserved region upstream of HDAg (Coller et al., 2018) was selected and adapted for use on the cobas6800. The lower limit of detection (LOD) was determined using a dilution panel of the HDV WHO standard (n = 21/dilution). Linearity and inclusivity were tested by preparing 10-fold dilution series (GT1-7 cell culture derived; GT8 patient derived; n = 5/dilution). Patient serum samples containing a variety of bloodborne viral pathogens were subjected to the assay to confirm exclusivity (n = 60).

Results LOD of the HDV Utility-Chanel assay (HDV_UTC) was determined as 3.86 IU/ml (95 %-CI: 2.95-5.05 IU/ml) with a linear range from 10-10^8 IU/ml (GT1). A linear relationship was observed for all 8 genotypes with slopes ranging from -3.422 to -3.783 cycles/log and an R2 range from 0.984-0.997 (for GT 5-7 results are pending). Inter-run and intra-run variability was 0.3 and 0.6 Ct (3xLOD), respectively. No false-positive results occurred. To evaluate clinical performance, serum samples of 110 anti-HDV-Ab positive patients were analyzed using the HDV_UTC and the CE-IVD RoboGene assay. 58/110 and 49/110 samples were concordant positive or negative, respectively. 3 samples (all Ct > 35.6) were only positive on the HDV_UTC assay (overall agreement 97,3 %). Quantitative comparison demonstrated a strong correlation (r: 0.922; 95 %-CI: 0.869-0.954; p-value: < 0.0001).

Conclusion We established a new quantitative and sensitive HDV PCR on the cobas6800 system. The use of highly automated, sample-to-result solutions for molecular diagnostics holds many inherent benefits over manual workflows, including improved reliability and reproducibility, as well as dynamic scaling of testing capacity. The assay showed excellent analytical and clinical performance, with inclusivity for all eight genotypes and a limit of quantification of 10 IU/ml, making it a sensitive new tool for HDV screening and viral load monitoring.

Publication History

Article published online:
04 January 2021