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Accurate quantification of HDV-RNA is essential for diagnosing and managing chronic hepatitis Delta (CHD), but there is considerable variability between different assays. In this study, we compared three methods for HDV-RNA quantification in both untreated and Bulevirtide (BLV)-treated CHD patients. Frozen plasma samples from untreated and BLV–treated CHD patients were tested in a single-center retrospective study by 3 different assays: Robogene HDV-RNA Quantification Kit 2.0 [Roboscreen GmbH; Lower Limit of Detection (LOD) <6 IU/mL, Lower Limit of Quantification (LOQ) 60 IU/mL on 7500 Fast Real-Time PCR System (Applied Byosystem)], EurobioPlex HDV PCR quantitative EBX071 [Eurobio Scientific, LOD <20 UI/mL, LOQ 50 UI/mL on CFX96™ real-time PCR detection system (Bio-Rad)] and AltoStar HDV RT-PCR RUO Kit 1.5 (Altona Diagnostics, estimated LOD <10 IU/mL estimated LOQ 20 UI/mL on AltoStar®AM16). 112 plasma samples from 79 CHD (10 untreated and 69 BLV-treated) patients were studied by 3 different assays (total 336 tests). All patients were HDV genotype 1 (available in 70). Median HDV RNA was 3.05 (-0.04-6.69) Log IU/mL with Robogene 2.0, 3.78 (-0.83-7.78) IU/mL with EurobioPlex EBX071, 3.22 (-0.31-7.03) IU/mL with AltoStar (Robogene vs. EurobioPlex p=0.005; Robogene vs. AltoStar p<0.0001; EurobioPlex vs. AltoStar p=0.5; overall p<0.0001). Compared to Robogene 2.0, EurobioPlex EBX071 reported higher (>0.5 Log IU/mL) HDV RNA levels in 33% samples, similar levels (<±0.5 Log IU/mL) in 55%. Likewise, compared to Robogene 2.0, AltoStar reported higher HDV RNA levels in 18% of positive samples, similar levels in 82%. Robogene tested target not detected (TND) or

A reliable quantification of serum hepatitis D virus (HDV) RNA is of paramount importance for a proper monitoring of patients under antiviral therapy. This quality-control study compares the diagnostic performances of quantitative HDV-RNA assays, used in clinical-practice. Two HDV-RNA sample panels were quantified at 30 laboratories by 6 commercial assays: RoboGene(N=9 laboratories), Eurobio/EliTech-platform(N=7), Altona(N=5), Anatolia(N=3), DiaPro(N=2), Nuclear-Laser-Medicine(N=1) and 3 in-house assays. Panel-A and -B comprised 8 serial dilutions of WHO/HDV standard(range:5-0.5logIU/ml) and 20 clinical samples(range:6-0.5logIU/ml), respectively. Laboratories quantified dilutions of Panel-A and -B 9 and 5 times, respectively, in order to define for each assay: i)sensitivity by 95%LOD(limit of detection), ii)precision by intra- and inter-run CV(coefficient of variation), iii)accuracy by the differences between expected-observed HDV-RNA loads, iv)linearity by linear-regression analysis. In Panel-A, 95%LOD varied across the assays underlining heterogeneous sensitivities: Altona had the lowest median 95%LOD (10[min-max:3-316]IU/ml), followed by Robogene (31[3-316] IU/ml), Nuclear-Laser-Medicine (31IU/ml) and EliTech (100[100-316]IU/ml). Moreover, 5 assays (Robogene/EliTech/Altona, Nuclear-Laser-Medicine/In-house) showed <0.5logIU/ml differences between expected and observed HDV-RNA for all dilutions while the remaining assays had HDV-RNA underestimations>1logIU/ml. In Panel-B, Altona and EliTech had the highest precision (mean intra-run CV<20%), followed by Robogene and Nuclear-Laser-Medicine (<30%). Inter-run CV was higher for all assays with only Altona, Nuclear-Laser-Medicine and EliTech maintaining this parameter<25%. Five assays (Robogene/Altona/EliTech/Nuclear-Laser-Medicine/In-house) showed a good linearity(R2>0.9) between LLOQ and 6.7logIU/ml. Conversely, a linearity drop emerged for HDV-RNA<1000IU/ml, with only Altona and Robogene retaining R2>0.85. This study underlines heterogeneous sensitivities (inter- and intra-assays), that could hamper the proper HDV-RNA quantification, particularly at low viral-loads. This raises the need to improve the diagnostic performance of most assays for properly identifying virological response to anti-HDV drugs. [ABSTRACT FROM AUTHOR]

Bulevirtide (BLV) was approved by EMA for CHD treatment, however no data exist about intrahepatic features of BLV-treated patients who underwent liver transplantation (LT). Aim of the study was to compare intrahepatic and clinical features of untreated and BLV-treated CHD patients who received LT. BLV-treated (SAVE-D study) and untreated CHD patients who underwent LT were compared. Clinical and virological features were collected at LT; HDV RNA was quantified with Robogene® 2.0. Intrahepatic (i) HDAg, HBsAg, and HBcAg staining were performed by immunohistochemistry (Ventana Benchmark Ultra System). 24 LT were studied, 14 untreated patients and 10 BLV-treated patients [14 (5-18) months therapy]. BLV-treated and untreated patients were similar in term of age at LT (56 vs. 49 years), male gender (60% vs. 29%), indication for LT (HCC 50% vs. 50%), HBsAg levels (3.6 vs. 3.8 LogIU/mL), NUC therapy (100%) and undetectable HBV DNA (100%). By contrast, BLV-treated patients had lower MELD (10 vs. 16, p=0.01), lower ALT (41 vs. 59, p=0.04) and lower HDV RNA (3.4 vs 5.2 LogIU/mL, p=0.08). At histology, iHBcAg stained negative in all patients, while iHBsAg stained negative in 2 (8%), 10-30% positive in 7 (29%), >30% in 15 (63%), without differences between groups. iHBsAg levels correlated with serum HBsAg (p=0.01), but not with serum HDV RNA. iHDAg stained negative in 2 patients (8%), 1-5% positive in 12 (50%), 10-50% in 7 (29%) and >50 in 3 (13%), without significant differences between groups, diffuse (46%) and focal (29%) being the most prevalent patterns. iHDAg correlated with serum HDV RNA (p=0.07), but not with serum HBsAg or iHBsAg. In CHD patients undergoing LT, iHBsAg was positive in most patients and correlated with serum HBsAg, while iHDAg staining was low and poorly correlated with serum HDV RNA, independently of BLV-treatment. [ABSTRACT FROM AUTHOR]

Serum HBV RNA and HBcrAg levels have been proposed as useful biomarkers in the management of HBV patients, however their role in chronic hepatitis Delta (CHD) is currently unknown. Consecutive untreated CHD patients were enrolled in a cross-sectional study in three EU centers. Clinical and virological characteristics were collected. Serum HBV RNA and HBcrAg levels were quantified by an automated real-time investigational assay (Cobas® 6800, Roche Diagnostics, Pleasanton, Ca, USA) and by LUMIPULSE® G HBcrAg assay (Fujirebio Europe), respectively. In 18 patients with available liver biopsies, intrahepatic analyses were performed. Overall, 240 HDV patients were enrolled: median age 46 years, 62% males, 53% cirrhotics, 57% NUC-treated, median ALT 70 U/L, HBsAg 3.8 log 10 IU/mL, 88% HBeAg-negative, median HDV RNA 4.9 log 10 IU/mL. HBV RNA tested positive (>10 cp/mL) in only 8% of the patients [median 40 (13-82,000) cp/mL], whereas HBcrAg was ≥3 log 10 U/mL in 77% [median 4.2 (3.0-8.0) log 10 U/mL]. By combining these biomarkers, 3 categories were identified: 23% double negative (HBV RNA neg/HBcrAg neg), 9% double positive (HBV RNA pos/HBcrAg pos) and 68% HBV RNA negative/HBcrAg positive. HBV RNA levels positively correlated with male sex and detectable HBV DNA, while positive HBcrAg correlated with higher HBsAg levels. Double positive patients were younger, non-European, with elevated ALT and HDV RNA levels and detectable HBV DNA. Intrahepatic HDV RNA and HBV RNA were positive in most samples, while intrahepatic levels of covalently closed circular (ccc)DNA were low. In untreated CHD, most patients had undetectable HBV RNA but quantifiable HBcrAg ("divergent pattern") in the absence of HBeAg. Additional studies aimed to unravel the molecular mechanisms underlying these findings are warranted. [ABSTRACT FROM AUTHOR]