TABLE 4.

Challenges and recommendations for the development of quantitative HDV RNA assays

Challenges	Recommendations
HDV RNA sequence variability
Primary sequence and secondary structures vary considerably between and within genotypes. There are replicative and defective quasispecies in clinical samples	Primer/probe design for RT-PCR assays should focus on highly conserved regions, and assay validation should assess geographically and temporally diverse clinical isolates (eg, at least 10–20 isolates), rather than cDNA or IVT RNA which do not have the same degree of secondary structure
Sequence data of HDV are limited (especially non-HDV-1), complicating the primer/probe design for RT-PCR assays	HDV sequencing should be performed consistently in clinical trials and epidemiological studies to increase the available sequence data
Long-term studies may be confounded by natural sequence variation, potentially impacting primer/probe binding affinity	Longitudinal studies should assess the primer/probe binding regions over time to monitor for sequence changes
Assay platforms and validation
Different assays with different performance characteristics are used across laboratories and trials	Assays should use the WHO international standard for validation, and an internal RNA control of known concentration at the RNA extraction stage, with primers/probe distinct from those used for HDV RNA. Additional cross trial/lab studies should be performed to allow comparison of results from clinical trials and to provide guidance for the use of these assays for patient management. Clinical trials and patient management should use a central laboratory with a validated assay (FDA approved/CE marked if available)
Assay performance data in non-HDV-1 are limited and difficult to generate given scarcity of non-HDV-1 samples	Non-HDV-1 in vitro transcribed RNA can be used for assay characterization, with the caveat that it lacks the secondary structures associated with viral RNA and therefore may have limited accuracy with respect to assay sensitivity and linearity. A repository of non-HDV-1 samples for assay characterization and validation would be valuable to the scientific community
Manual RNA extraction has been reported to be more sensitive than automated procedures but is prone to higher variability and is more labor intensive	Automated assays, ideally on standard platforms, should be developed/used as much as possible
Data interpretation
Use of assays with different performance characteristics complicates data interpretation	Data should be reported in IU/mL. When selecting an assay platform, the sensitivity and specificity should be reported, and preferably these should be comparable to the best performing assays available
Clinical relevance of undetectable vs. detectable HDV RNA is uncertain	Clinical studies are needed to assess threshold of HDV RNA for long-term clinical outcomes (suppressive therapy) and/or viral relapse (finite therapy)
Different ways to report HDV RNA values below LLoQ are used	Use consistent nomenclature to report HDV RNA values below the quantitative range (below LLoQ): ie, data should be reported as either below LLoQ, target detected or below LLoQ, target not detected. Use of reporting below LoDa should be avoided because it incorrectly implies virus absence
For novel treatments, there is no clear guidance/consensus on frequency of HDV RNA testing during treatment and during follow-up	Guidelines will need to be developed considering resource-limited regions

^aLimit of detection (LoD) is defined as the lowest concentration at which ≥95% of replicates test positive.

Abbreviations: CE, conformité européenne; FDA, Food and Drug Administration; IVT, in vitro transcription; LLoQ, lower limit of quantitation; LoD, limit of detection; RT-PCR, real-time PCR; WHO, World Health Organization.