Early Dynamics of Hepatitis B Virus (HBV)-DNA and Surface Antigen (HBsAg) in Ramp-Up Phase of Viremia: Implications for Performance Evaluation of Blood Screening Assays

The Common Specifications/EU 2017/746 regulation for market approval of class D in vitro diagnostic devices (IVDs) intended for detection of blood borne viruses requires testing of the International Standard and 10-30 seroconversion panels to demonstrate 'state of the art' assay performance. We examined whether these requirements for performance evaluation are reasonable for HBV-DNA and HBsAg assays. For this purpose, we quantified HBsAg and HBV-DNA (genotype A) in the ramp-up phase of five seroconversion panels and demonstrated a remarkably parallel increase in the Log concentration of both analytes over time. Testing of seroconversion panels by three nucleic acid amplification technology (NAT) methods in multiple replicates and probit analysis with sufficient critical samples from all five panels taken together showed detection limits in copies/mL that were comparable to those on a HBV-DNA genotype A standard dilution panel. This indicates that the viral doubling time in the ramp-up phase is equal above and below the quantification limit of the viral load assay. The geometric mean HBsAg (PRISM) cutoff crossing point was 20 days later than the 50% NAT (Ultrio Plus) conversion point equivalent to 1500 (range: 1100-2200) and 4.8 (CI: 3.7-6.4) HBV-DNA copies/mL, respectively. Analytical sensitivity data of different NAT assay versions obtained over a decade demonstrated that the detection limit on the International Standard is not representative of all genotyped reference samples. From our detailed mathematical analysis, we conclude that HBV-DNA and HBsAg standard dilution series are functionally equivalent to seroconversion panels. A general requirement of a 95% detection limit <= 100 HBV-DNA copies/mL for different viral genotypes would be a better-defined regulation for EU market approval of NAT blood screening assays than the testing of multiple seroconversion panels to claim 'state of the art' performance.

Automated summary

This study examined whether the performance evaluation requirements for HBV-DNA and HBsAg assays are reasonable and proposed a better-defined regulation for NAT blood screening assays in the EU market.