HDV RNA assays: Performance characteristics, clinical utility, and challenges

Abstract

Co-infection with hepatitis B virus (HBV) and hepatitis D virus (HDV) results in hepatitis D, the most severe form of chronic viral hepatitis, frequently leading to liver decompensation and hepatocellular carcinoma. Pegylated interferon alpha, the only treatment option for chronic hepatitis D for many years, has limited efficacy. New treatments are in advanced clinical development, with one recent approval. Diagnosis and antiviral treatment response monitoring are based on detection and quantification of HDV RNA. However, the development of reliable HDV RNA assays is challenged by viral heterogeneity (at least 8 different genotypes and several subgenotypes), intra-host viral diversity, rapid viral evolution, and distinct secondary structure features of HDV RNA. Different RNA extraction methodologies, primer/probe design for Nucleic Acid Tests, lack of automation, and overall dearth of standardization across testing laboratories contribute to substantial variability in performance characteristics of research-based and commercial HDV RNA assays. A WHO standard for HDV RNA, available for about 10 years, has been used by many laboratories to determine the limit of detection of their assays and facilitates comparisons of RNA levels across study centers. Here we review challenges for robust pan genotype HDV RNA quantification, discuss particular clinical needs and the importance of reliable HDV RNA quantification in the context of drug development and patient monitoring. We summarize distinct technical features and performance characteristics of available HDV RNA assays. Finally, we provide considerations for the use of HDV RNA assays in the context of drug development and patient monitoring.

Hepatitis D

“…The performance of diagnostic tests for HDV is highly variable. Because of the high-sequence heterogeneity of the HDV genome, some HDV-RNA assays may yield false-negative results in up to 5% of samples or underestimate viral load with certain genotypes, and up to a 2-log 10 (100-fold) difference in quantitative results have been reported when the same sample was tested in different laboratories …”

HDV screening in chronic HBV: An unmet need of growing importance

“…However, it is important to remember that detection of HDV infection requires accurate, reliable, and standardized diagnostic tests before universal screening can be implemented. Currently, there is significant variability in the performance of anti-HDV antibody tests and none are Food and Drug Administration (FDA) approved for diagnostic testing 10 . In addition, HDV-RNA assays have not yet been validated and have variable lower limits of quantification and detection.…”

“…In addition, HDV-RNA assays have not yet been validated and have variable lower limits of quantification and detection. Finally, the sensitivity and specificity of HDV-RNA assays for isolates containing the 8 distinct genotypes of HDV have not been established 10,11 …”

“…Anti-HDV serological testing helps identify those with acute versus chronic and resolved infections. Patients with acute HDV-HBV coinfection typically have anti-HDV IgM detectable for 1–3 months after exposure in the serum and HDV-RNA 2,10 . In contrast, a detectable anti-HDV IgG serological test is evidence of either past or current infection, and active HDV infection is defined by detectable HDV-RNA in the blood (Figure 1).…”