HBV‐RNA Co‐amplification May Influence HBV DNA Viral Load Determination

Abstract

Despite effective hepatitis B virus (HBV)-DNA suppression, HBV RNA can circulate in patients receiving nucleoside/ nucleotide analogues (NAs). Current assays quantify HBV DNA by either real-time polymerase chain reaction (PCR), which uses DNA polymerase, or transcription-mediated amplification, which uses reverse-transcriptase (RT) and RNA polymerase. We assessed the effect of RT capability on HBV-DNA quantification in samples from three cohorts, including patients with quantified HBV RNA. We compared the HBV-DNA levels by real-time PCR (cobas HBV, Roche 6800/8800; Xpert HBV, Cepheid), transcription-mediated amplification (Aptima HBV, Hologic), and real-time PCR with added RT capability (cobas HBV+RT). In the first cohort (n = 45) followed over 192 weeks of NA therapy, on-treatment HBV-DNA levels were higher with cobas HBV+RT than cobas HBV (mean difference: 0.14 log 10 IU/ mL). In a second cohort (n = 50) followed over 96 weeks of NA therapy, HBV-DNA viral load was significantly higher with the cobas HBV+RT and Aptima HBV compared with the cobas HBV test at all time points after initiation of NA therapy (mean difference: 0.65-1.16 log 10 IU/mL). A clinically significant difference was not detected between the assays at baseline. In a third cohort (n = 53), after a median of 2.2 years of NA therapy, we detected HBV RNA (median 5.6 log 10 copies/mL) in 23 patients (43.4%). Median HBV-DNA levels by Aptima HBV were 2.4 versus less than 1 log 10 IU/mL in samples with HBV RNA and without HBV RNA, respectively (P = 0.0006). In treated patients with HBV RNA, Aptima HBV measured higher HBV-DNA levels than Xpert HBV and cobas HBV. Conclusion:Tests including an RT step may overestimate HBV DNA, particularly in samples with low viral loads as a result of NA therapy. This overestimation is likely due to amplification of HBV RNA and may have an impact on clinical decisions. (Hepatology Communications 2020;4:983-997).

Hepatitis B Virus RNA Detection and a Mindful Use of Serum Hepatitis B Virus DNA and Hepatitis B Surface Antigen Measures in Clinical Practice

“…The presence of serum HBV RNA should not interfere with HBV DNA detection, but it depends on when (on or off NA treatment) and how (which assay is issued) HBV DNA is measured. This is consistently shown by Maasoumy et al (10) in this issue of Hepatology Communications in their excellent methodology manuscript that addresses the issue of the specificity of quantitation of circulating HBV DNA by nucleic acid amplification techniques, which use different amplification strategies. The authors demonstrated that the inclusion of a reverse transcriptase step in the amplification process causes an overestimation of HBV DNA copies due to the detection of circulating HBV RNA.…”

“…In conclusion, both manuscripts (6,10) in this issue of Hepatology Communications provide compelling evidence that emphasizes once again the clinical importance of the mandatory role of laboratory medical experts who help to identify the more appropriate assays needed to address specific clinic-pathologic questions of medical practice, such as the ones raised by monitoring the response to NAs.…”

Standardized Hepatitis B Virus RNA Quantification in Untreated and Treated Chronic Patients: a Promising Marker of Infection Follow-Up

“…The lower limit of quantitation (LLOQ) of this test is 10 IU/mL, and the LOD for plasma samples is 6.6 IU/mL ( 21 ). Of note, this assay enables viral DNA quantification without any overestimation due to the presence of circulating HBV RNA because it lacks a retro-transcription step ( 36 ). Samples with detectable DNA results below the LLOQ were assigned an arbitrary value of 0.5 log 10 IU/mL.…”

“…Circulating HBV RNA levels were measured with the Cobas HBV RNA real-time quantitative RT-PCR assay for use on the Cobas 6800/8800 Systems (Roche) Molecular Systems ( 36 , 37 ). This assay has an LLOQ of 10 copies/mL and a linear range of 10 to 10 9 copies/mL on armored RNA ( 37 ).…”

Rapid Diagnostics for Hepatitis B and C Viruses in Low- and Middle-Income Countries

“…Assessment of the platform within a tertiary care hospital clinic in Addis Ababa, Ethiopia, demonstrated utility of the Xpert HBV VL to identify patients requiring initiation of treatment, with an Xpert cutoff of 4,190 IU/mL and 35,800 IU/mL equivalent to ≥ 2,000 IU/mL and ≥20,000 IU/mL, as determined by a high-throughput LBT method (79). A strong positive correlation in quantitative results among most platforms was observed (74,(80)(81)(82)(83), suggesting the Xpert HBV VL assay is a promising alternate to conventional high throughput platforms for initiating and monitoring of antiviral treatment, although the cost-effectiveness of this would be contingent upon sample throughput.…”