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

Abstract

The measurement and interpretation of HBV DNA and RNA levels in HBV infected patients treated with antiviral therapy supports the objective of HBV disease management. Here, we quantified circulating HBV RNA through a standardized and sensitive assay in follow-up samples from both naive and treated patients as a marker of infection evolution. HBV DNA (HBV DNA for use in Cobas 6800/8800 Automated Roche Molecular Systems), RNA (Roche HBV RNA Investigational Assay for use in the Cobas 6800/8800; Roche), HBeAg and HBsAg (Elycsys HBsAg chemiluminescence immunoassay by Cobas 8000; Roche), and core-related antigen (Lumipulse G chemiluminescence assay; Fujirebio) levels were measured in cohorts of untreated or nucleos(t)ide treated, HBV-infected subjects in an outpatient hospital setting. HBV DNA levels in untreated people were 3.6 log₁₀ higher than corresponding RNA levels and were stable over 5 years of observation. While only five of 52 treated patients had DNA levels below the lower limit of quantification (10 IU/mL) at the end of follow-up, 13 had HBV RNA levels persistently above this limit, including eight with undetectable DNA. In samples with undetectable core-related antigen we observed a median HBsAg titer 2.7-fold higher than in samples with undetectable RNA (adjusted P = 0.012). Detectable HBV RNA with undetectable HBV DNA was a negative predictor of HBsAg decrease to a level ≤100 IU/mL (P = 0.03). In naive patients the difference between HBV DNA and RNA was higher than previously reported. HBV RNA rapidly decreased during treatment. However, in some cases, it was detectable even after years of effective therapy, being a negative predictor of HBsAg decrease. The investigational RNA assay for use on the Cobas 6800/8800 instruments is a sensitive and standardized method that could be applied in general management of HBV infection. IMPORTANCE This study focused on the quantification of circulating HBV RNA by using a standardized and sensitive assay. Thanks to this system we observed a higher difference between circulating HBV DNA and RNA than previously reported. In treated patients, HBV RNA decreased together with DNA, although some patients presented detectable levels even after years of successful antiviral treatment, suggesting a persistent viral transcription. Of note, the detection of viral RNA when HBV DNA is undetectable was a negative predictor of HBsAg decrease to a level ≤100 IU/mL. This assay could be extremely helpful in HBV patients management to study viral transcription and to identify those treated patients that may achieve sustained viral suppression.

Keywords: HBV; HBV-RNA; RT-PCR; antiviral treatment; biomonitoring.

FIG 1

HBV DNA and HBV RNA trends over time in naive patients. HBV DNA (black open circles) and HBV RNA (gray open squares) titers (log₁₀ copies/mL) throughout follow-up in treatment-naive patients. The number of patients (N) at each time point is shown in italics above the x-axis.

FIG 2

HBV RNA and HBV DNA during follow-up in NA-treated patients. The graphs show HBV RNA and DNA titers (log₁₀ copies/mL) during years of treatment. Patients in group A ([A and C] for viral RNA and DNA, respectively) had HBV RNA in on-treatment samples below the lower limit of quantification (LLOQ), except for isolated blips in three patients. Patients in group B ([B and D] for viral RNA and DNA, respectively) had HBV RNA levels > LLOQ except for an isolated detectable but < LLOQ result for one patient. Patients with RNA < LLOQ at baseline and all on-treatment time points (n = 20) are not shown.

FIG 3

HBsAg titer in patients with undetectable HBcrAg or HBV RNA. The plot shows the HBsAg titer in those treated patients with undetectable HBcrAg (≤2.5 log₁₀ U/mL) or undetectable HBV RNA (<LOD). Only those samples with both markers analyzed were included. Horizontal bars represent the median and interquartile range.