Assessment of the cobas® HBV RNA investigational assay in the setting of nucleoside analog therapy cessation.

HBV RNA is used as a marker of cccDNA transcription and is applicable in the setting of nucleos(t)ide analog (NA) treatment, which suppresses HBV DNA. Traditional assays for quantification of HBV RNA rely on labor-intensive 3'RACE assays targeting the polyA tail. In this study, the high-throughput Roche cobas®HBV RNA investigational assay was assessed on the Roche cobas® 6800 automated platform. Of 969 samples collected for a NA treatment cessation trial, and tested on the cobas assay, 249 were analyzed for sensitivity, reproducibility, sample type applicability, and results were compared to a RACE-based assay. Results of 97 paired serum and plasma samples demonstrated an excellent correlation of 0.98. However, 14.5% of plasma samples yielded detectable (below the limit of quantification) results, when the paired serum was undetectable, and plasma was shown to yield a statistically significant (p < 0.001) greater mean 0.119 log10 copies/ml. Quantification of 152 samples showed good correlation (0.91) between the cobas and RACE assays. The cobas assay demonstrated superior lower limit of quantification, 10 copies/ml, which resulted in detection of 13.2% more samples than the RACE assay. Reproducibility and linear range of the automated assay were also confirmed. The Roche cobas assay for HBV RNA is sensitive and highly recommended.

ITPA genotype protects against anemia during peginterferon and ribavirin therapy but does not influence virological response.

UNLABELLED: On-treatment anemia is associated with higher sustained virological response (SVR) rates during peginterferon plus ribavirin (RBV) therapy. Inosine triphosphatase (ITPA) variants causing ITPase deficiency have been shown to protect against RBV-induced anemia. However, ITPase activity has not been associated with SVR. To study this discrepancy, we examined the relationships between ITPase activity, on-treatment anemia, SVR, and RBV levels in hepatitis C virus genotype 1 (HCV-1) patients from the CHARIOT study. ITPA genotype (rs7270101, rs1127354) was used to define ITPase activity in 546 patients. Plasma RBV levels were measured using high-performance liquid chromatography (HPLC). Relationships between ITPase activity, on-treatment hemoglobin (Hb) levels, RBV levels, and SVR were tested using regression modeling, survival analysis, and locally weighted scatterplot smoothing (LOWESS) plot analysis. Hb decline was independently associated with SVR (P<0.0001). ITPase deficiency was present in 35%. ITPase deficiency strongly protected against Hb decline (P<0.0001), but was not associated with SVR (P=0.28). The probability of SVR increased with lower nadir Hb for both wild-type and deficient ITPase activity, but the association curve shifted to describe a parallel relationship at higher Hb levels in patients with ITPase deficiency. In a subset (n=203), we tested the hypothesis that the association between Hb decline and SVR reflected RBV levels rather than actual Hb level. RBV levels were associated with on-treatment Hb decline and SVR, but not ITPase activity. In regression models, adjustment for RBV levels attenuated the association between Hb decline and SVR. CONCLUSION: ITPase deficiency protects against RBV-induced anemia, but is not associated with SVR. Our data suggest that the relationship between Hb decline and SVR is not mechanistic, but is linked to RBV levels.

IL28B genotype is not useful for predicting treatment outcome in Asian chronic hepatitis B patients treated with pegylated interferon-α.

BACKGROUND AND AIM: IL28B genotype predicts response to pegylated interferon (peg-IFN)-based therapy in chronic hepatitis C. However, the utility of IL28B genotyping in chronic hepatitis B (CHB) cohorts treated with peg-IFN is unclear. It was investigated whether IL28B genotype is associated with peg-IFN treatment outcomes in a predominantly Asian CHB cohort. METHODS: This was a retrospective analysis of CHB patients treated with 48 weeks of peg-IFN monotherapy. IL28B genotype (rs12979860) was determined (TaqMan allelic discrimination kit). Baseline hepatitis B virus (HBV)-DNA, alanine aminotransferase, and liver histology were available. The primary end-points were HBV e antigen (HBeAg) seroconversion with HBV-DNA < 2000 IU/mL 24 weeks post-therapy (HBeAg-positive patients) and HBV-DNA < 2000 IU/mL 24 weeks after peg-IFN (HBeAg-negative patients). The association between IL28B genotype and peg-IFN outcomes was analyzed. RESULTS: IL28B genotype was determined for 96 patients. Eighty-eight percent were Asian, 62% were HBeAg positive, and 13% were METAVIR stage F3-4. Median follow-up time was 39.3 months. The majority of patients carried the CC IL28B genotype (84%). IL28B genotype did not differ according to HBeAg status. The primary end-points were achieved in 27% of HBeAg-positive and 61% of HBeAg-negative patients. There was no association between IL28B genotype and the primary end-point in either group. Furthermore, there was no difference in HBeAg loss alone, HBV surface antigen, alanine aminotransferase normalization, or on-treatment HBV-DNA levels according to IL28B genotype. CONCLUSIONS: In the context of a small possible effect size and high frequency in Asian populations, IL28B genotyping is likely to have, at best, limited clinical utility for predicting peg-IFN treatment outcome for CHB patients in the Asia-Pacific region.

Baseline serum HBV RNA is associated with the risk of hepatitis flare after stopping nucleoside analog therapy in HBeAg-negative participants.

BACKGROUND AND AIMS: HBV RNA in peripheral blood reflects HBV cccDNA transcriptional activity and may predict clinical outcomes. The prospective Melbourne HBV-STOP trial studied nucleot(s)ide analog discontinuation in HBeAg-negative non-cirrhotic participants with long-term virological suppression. Ninety-six weeks after stopping treatment, the proportion of participants with virological relapse (HBV DNA > 2000 IU/mL), biochemical relapse (ALT > 2 × ULN and HBV DNA > 2000 IU/mL), or hepatitis flare (ALT > 5 × ULN and HBV DNA > 2000 IU/mL) was 89%, 58%, and 38%, respectively. We evaluated the ability of serum HBV RNA levels to predict these outcomes. APPROACH RESULTS: HBV RNA levels were measured using the Roche cobas 6800/8800 HBV RNA Investigational Assay. Sixty-five participants had baseline and longitudinal off-treatment specimens available for RNA testing. HBV RNA was detectable at baseline in 25% of participants and was associated with a higher risk of biochemical relapse (81% vs. 51%, p value 0.04) and hepatitis flare (63% vs. 31%, p value 0.04). Participants who had undetectable serum HBV RNA as well as HBsAg ≤ 100 IU/mL at baseline were less likely to experience virological relapse (4 of 9, 44%) than participants with detectable HBV RNA and HBsAg level > 100 IU/mL (15/15, 100%; p value 0.0009). Off-treatment levels of HBV RNA were correlated with HBV DNA and were associated with the risk of hepatitis flare. CONCLUSIONS: Serum HBV RNA may be a useful biomarker for guiding clinical decision-making before stopping nucleot(s)ide analog therapy. Baseline HBV RNA and HBsAg levels are associated with the risk of clinical relapse, hepatitis flare, and disease remission off-treatment.

Autochthonous and Travel Acquired Hepatitis E Virus in Australia.

BACKGROUND: Hepatitis E virus (HEV) is a common cause of acute viral hepatitis with significant morbidity and mortality, particularly in pregnant women. There are four major genotypes which can cause disease in humans. Genotypes 1 and 2 are usually associated with outbreaks and spread via facal/oral route or contaminated water. Genotypes 3 and 4 are zoonotic and usually associated with handling of pigs or consumption of contaminated pork. The strains circulating in Australia have never been characterized. RATIONALE/AIMS: The aims for this project are to identify the HEV genotypes found in Australia and link them to possible sources of transmission by phylogenetic analysis. MATERIALS AND METHODS: Between 2015 and 2020, 91 HEV isolates were sequenced and genotyped using an in-house PCR. Sixty-six of these were also sequenced by using the international HEVnet primers. Genotypes were determined using the BLASTn program. Relatedness to other strains in Australia was determined by phylogenetic analyses of the HEVnet sequences. Isolates were also stratified by state of origin, gender, age, predisposing factors and travel history (if known). RESULTS: Of the 91 HEV isolates sequenced, 55 (60.4%) were genotype 1. There were 34 (37.4%) genotype 3 strains and two genotype 4 (2.2%). At least 20 of the genotype 1 strains have been linked to travel in India, and another three with Pakistan. Five of the "Indian" strains were closely related and are suspected to have originated in Gujarat. Phylogenetic analysis also showed that 12 genotype 3 strains were genetically related and potentially acquired in/from New South Wales, Australia. The two genotype 4 strains may have originated in China. DISCUSSION: This is the first study to describe the HEV isolates identified in Australia. The results infer that HEV may be acquired during overseas travel as well as locally, presumably from consumption of pork or pork-related products. The phylogenetic analyses also reveal clusters of infection originating from India and Pakistan. This study provides some insight into the source and epidemiology of HEV infection in Australia which may be used to guide public health procedure and enable the implementation of measures to deal with potential outbreaks of infection.

Multicenter clinical evaluation of alinity m HBV assay performance.

BACKGROUND: Accurate molecular methods to detect and quantify hepatitis B virus (HBV) DNA are essential to diagnose chronic infections, guide treatment decisions, assess response to treatment, and determine risk of HBV-related complications. New generations of real-time HBV DNA assay platforms provide results in less than 2-3 h, with continuous loading of specimens and true random-access capability. OBJECTIVES: We examined the clinical performance of the new Alinity m HBV assay, run on the fully automated, continuous, random-access Alinity m platform, to accurately detect and quantify HBV DNA in a large series of patient samples infected with different HBV genotypes frequently encountered in clinical practice. STUDY DESIGN: This international, multisite study assessed the precision and reproducibility of the Alinity m HBV assay and compared its performance to four HBV assays currently in clinical use. RESULTS: The Alinity m HBV assay demonstrated linear quantitation of HBV DNA in plasma samples, with high precision (coefficient of variation 4.1 %-8.8 %) and reproducibility. The Alinity m HBV assay showed excellent correlation (correlation coefficients ≥0.947) with comparator HBV assays, with an overall observed bias ranging from -0.07 to 0.17 Log10 IU/mL. 97 % of quantifiable patient results were <1 Log10 IU/mL different than the respective comparator assays, with comparable results across HBV genotypes. CONCLUSIONS: The newly developed real-time PCR-based Alinity m HBV assay is sensitive, reproducible, and accurately quantifies HBV DNA levels from HBsAg-positive patients across the full dynamic range of quantification.

Multicenter clinical evaluation of alinity m HCV assay performance.

BACKGROUND: Nucleic acid testing is essential for the detection and quantification of HCV RNA in the diagnosis of HCV infection and treatment monitoring. The Alinity m HCV assay was recently developed by Abbott Molecular for rapid detection and quantification of HCV RNA on the fully automated, continuous, random-access Alinity m analyzer. OBJECTIVES: Our study assessed the performance of the new Alinity m HCV assay for detection and quantification of HCV RNA in a large series of patient samples of various genotypes. This international, multicentric study evaluated the linearity, precision, and reproducibility of the Alinity m HCV assay and its performance in comparison to three other HCV assays currently used in clinical practice. RESULTS: The Alinity m HCV assay demonstrated high linearity (correlation coefficient r = 1.00), precision (coefficients of variation [CV] 6.6-13.5 %) and reproducibility (CV 1.7-4.3 % across three control lots). At a concentration near the lower limit of detection, the Alinity m HCV assay exhibited >98 % detectability. The Alinity m HCV assay showed excellent correlation with comparator HCV assays in serum (n = 406) and plasma (n = 1401) samples (correlation coefficients ≥0.96, bias 0.01 to 0.14 Log10 IU/mL). More than 95 % of the quantified results with the Alinity m HCV assay were less than mean bias ± 1.96 SD different from those of the comparator assays. CONCLUSIONS: The newly developed Alinity m HCV assay is sensitive, reproducible, and accurately quantifies HCV RNA levels in serum and plasma samples from patients with chronic HCV infection, with no impact of HCV genotype on assay performance.