Development and Evaluation of Diagnostic Methods for Hepatitis Delta Virus (HDV): Molecular and Serological Approaches

Abstract

Background & Aim: Detecting the hepatitis D virus (HDV) RNA via PCR is challenging due to the heterogeneity of genotypes (GT), high GC content (approximately 60%), and significant levels of intramolecular base pairings. HDV can self-replicate, but it relies on the hepatitis B virus (HBV) for viral assembly. Recent studies have shown, both in vitro and in a mouse model, that viruses other than HBV, such as the hepatitis C virus (HCV), can package HDV and facilitate the release of infectious particles. The aim of this dissertation was I) to establish and validate an RT-qPCR assay for the detection and quantification of all 8 HDV GT on a fully automated, high-throughput platform (cobas6800) and II) to evaluate HCV-mediated HDV propagation in HCV mono-infected patients in Germany. Methods: Previously published primers were adapted for use on a fully automated platform. Serial dilutions of the HDV WHO standard were employed to assess the technical performance of the assay (lower limit of detection, linearity, and precision). Inclusivity was evaluated using cell culture-derived viruses, and anti-HDV positive sera (n=323) were included to compare clinical performance with a CE-IVD assay. HCV PCR-positive and hepatitis B antigen-negative serum samples (n=110) were utilized to investigate HDV PCR and anti-HDV positivity in HCV mono-infected patients. Additionally, serology markers for HBV infection were determined. Results: The RT-qPCR assay demonstrated excellent technical and clinical performance, with a lower limit of detection of 3.86 IU/ml (95% CI: 2.95 – 5.05 IU/ml), inclusivity, and linearity for all HDV GT (slopes: -3.48 to -4.413; r² > 0.92), as well as high precision. A comparison with a CE-IVD assay revealed a strong correlation (r²: 0.87) and an overall agreement of 97.3%. The evaluation of HCV/HDV co-infection showed that no HDV PCR-positive patients could be identified (n=0/323) and all anti-HDV positive patients (n=8/316) exhibited signs of past but currently immune-controlled HBV infection (n=8/8 anti-HBc positive). Conclusion: In conclusion the excellent performance observed demonstrates that the HDV RT-qPCR assay can be used as a powerful tool for diagnosing active HDV infection and monitoring patients. Furthermore, the results suggest that the likelihood of HCV-mediated HDV infection without signs of previous HBV infection is low, at least in regions with low HDV prevalence.