HBV RNA as a Biomarker for Safe Antiviral Discontinuation: A Prospective Study of Nucleos(t)ide Analogue Withdrawal.

INTRODUCTION: Withdrawal of nucleos(t)ide analog (NA) therapy is associated with hepatitis B surface antigen (HBsAg) loss and sustained, off-therapy partial cure (normal ALT [≤30 U/L males, ≤20 U/L females) with HBV DNA <2000 IU/mL) but should be offered only to those most likely to benefit. HBVRNA may be useful for risk stratification. METHODS: The Hepatitis B Research Network Immune-Active Trial prospectively evaluated treatment with tenofovir (TDF) for 192 weeks ± peginterferon-α (PegIFN) for initial 24 weeks followed by protocolized withdrawal of TDF amongst eligible participants (NCT01369212). HBV RNA was evaluated as predictor of ALT flares and sustained partial cure (HBV DNA<2000 IU/mL) 48 weeks after TDF withdrawal. RESULTS: Of 93 participants discontinuing TDF (52 in TDF+PegIFN and 41 in TDF alone), 52 (55.9%) had unquantifiable HBVRNA at end-of-treatment. ALT flares (>5xULN) at 48 weeks off-therapy occurred in 33.3%, with pre-treatment age (≥35 years) and quantifiable HBV RNA at end-of-treatment, the best predictors (AUROC 0.74 and 0.85, training and test set) of ALT flare. A total of 26 (28.3%) had sustained partial cure; 3 (11.5%) with ALT flare. Non-quantifiable HBVRNA and qHBsAg <100 IU/mL at end-of-treatment were the best predictors of sustained partial cure (AUROC 0.84 and 0.93, training and test set). If HBVRNA was quantifiable at end-of-treatment, the likelihood of sustained partial cure was only 3% whereas if HBV RNA was unquantifiable and qHBsAg <100 IU/mL, this likelihood was 73%. CONCLUSIONS: HBVRNA is a useful biomarker in predicting likelihood of achieving sustained partial cure and safe withdrawal of NAs.

An evolutionary concept analysis: stigma among women living with hepatitis C.

BACKGROUND: Stigma is a complex social phenomenon that leads to marginalization and influences the course of illness. In the context of hepatitis C virus (HCV), stigma is a well-documented barrier to accessing care, treatment, and cure. In recent years, HCV rates among women have increased, resulting in an urgent need to address stigma and its harmful effects. The purpose of this concept analysis was to investigate stigma in the context of women living with HCV using Rodgers' evolutionary method. METHODS: PubMed, CINAHL, Scopus, Medline, PsycINFO, and Nursing and Allied Health were used to identify articles describing HCV stigma among women. Articles from peer-reviewed journals and geographic locations, published between 2002-2023, were included in the analysis. As specified in Rodgers' evolutionary method, articles were analyzed with a focus on the concept's context, surrogate and related terms, antecedents, attributes, examples, and consequences. RESULTS: Following screening, 33 articles were selected for inclusion in the analysis. Discrimination and marginalization were identified as surrogate and related terms to stigma; and antecedents of stigma were identified as limited knowledge, fear of diagnosis, and disclosure. Prevalent attributes of stigma in the literature were described as feelings of decreased self-worth, negative stereotyping, and fear of transmission. Importantly, HCV stigma among women is unique in comparison to other forms of infectious disease-related stigma, primarily due its impact on women's identity as mothers and caregivers. Stigmatization of women living with HCV resulted in negative consequences to personal relationships and healthcare access due to decreased health-seeking behaviours. Although access to HCV treatment has changed considerably over time, a temporal analysis could not be completed due to the limited number of articles. CONCLUSIONS: Stigma in the context of women living with HCV has its own unique antecedents, attributes, and consequences. This enhanced understanding of stigma among women living with HCV has the potential to inform improved and more effective approaches to care, which will be required to reach HCV elimination. Furthermore, this analysis identifies stigma layering and stigma in the direct-acting antiviral  treatment era as areas for more in-depth future inquiry.

Mixed-Genotype HCV Direct Acting Antiviral Outcomes: A CANUHC Analysis.

The prevalence of mixed hepatitis C virus (HCV) genotype infection in a representative Canadian HCV cohort is reported and virological response with direct acting antiviral (DAA) treatment was evaluated. 3272 HCV-positive participants were enrolled, of which 2945 (90.0%) initiated DAA therapy. 0.8% were identified with mixed genotype infection. Overall sustained virological response (SVR) was 99.1% and did not differ based on mixed genotype status. Any historical disadvantage to achieving cure with HCV treatment in mixed genotype infection has been overcome by current DAA regimens.

Differential Gene Expression in the Upper Respiratory Tract following Acute COVID-19 Infection in Ambulatory Patients That Develop Long COVID.

Background: Post-acute sequelae of COVID-19, or long COVID, is a condition characterized by persistent COVID-19 symptoms. As long COVID is defined by clinical criteria after an elapsed period, an opportunity for early intervention may aid in future prophylactic approaches; however, at present, the pathobiological mechanisms are multifactorial. By analyzing early virally infected upper respiratory tract tissue prior to eventual clinical diagnosis, it may be possible to identify biomarkers of altered immune response to facilitate future studies and interventions. Methods: This is a sub-group analysis of samples collected from those with confirmed COVID-19. RNA extraction from nasopharyngeal/mid-turbinate samples, sequencing, and bioinformatic analysis were performed to analyze long COVID and non-long COVID cohorts at day 14 post infection. Differences in mean viral load at various timepoints were analyzed as well as serological data. Results: We identified 26 upregulated genes in patients experiencing long COVID. Dysregulated pathways including complement and fibrinolysis pathways and IL-7 upregulation. Additionally, genes involved in neurotransmission were dysregulated, and the long COVID group had a significantly higher viral load and slower viral clearance. Conclusions: Uncovering early gene pathway abnormalities associated with eventual long COVID diagnosis may aid in early identification. We show that, post acute infection, in situ pathogenic deviations in viral response are associated with patients destined to meet consensus long COVID diagnosis that is entirely dependent on clinical factors. These results identify an important biological temporal window in the natural history of COVID-19 infection and long COVID pathogenesis amenable to testing from standard-of-care upper respiratory tract specimens.

Estimating the prevalence of hepatitis delta virus infection among adults in the United States: A meta-analysis.

BACKGROUND AND AIMS: Suboptimal awareness and low rates of hepatitis delta virus (HDV) testing contribute to underdiagnosis and gaps in accurate estimates of U.S. HDV prevalence. We aim to provide an updated assessment of HDV prevalence in the U.S. using a comprehensive literature review and meta-analysis approach. METHODS: A comprehensive literature review of articles reporting HBsAg seroprevalence and anti-HDV prevalence was conducted to calculate country-specific rates and pooled prevalence of CHB and HDV using meta-analyses. Country-specific CHB and HDV rate estimates were combined with number of foreign-born (FB) persons in the U.S. in 2022 from U.S. Census Bureau to estimate total numbers of FB with CHB and HDV, respectively. These estimates were further combined with updated estimates of U.S.-born persons with CHB and HDV to yield the total number of persons with CHB and HDV. RESULTS: In 2022, we estimated 1.971 million (M) (95% CI 1.547-2.508) persons with CHB; 1.547 M (95% CI 1.264-1.831) were FB and 0.424 M (95% CI: 0.282-0.678) were U.S.-born. The weighted average HDV prevalence among FB persons in the U.S. was 4.20% (64 938 [95% CI 33055-97 392] persons), among whom 45% emigrated from Asia, 25% from Africa, and 14% from Europe. When combined with updated estimates of U.S.-born persons with HDV, we estimate 75 005 (95% CI: 42187-108 393) persons with HDV in the U.S. CONCLUSIONS: Including both FB and U.S.-born persons, we estimated that 1.971 M and 75 005 persons were living with CHB and HDV, respectively, in the U.S. in 2022.

An updated assessment of hepatitis delta prevalence among adults in Canada: A meta-analysis.

Foreign-born (FB) persons represent a large proportion of adults with chronic hepatitis B (CHB) in Canada due to higher prevalence rates in countries of birth for FB persons. Suboptimal awareness and low rates of hepatitis delta virus (HDV) testing contribute to underdiagnosis and gaps in accurate estimates of Canada HDV prevalence. We aim to provide an assessment of CHB and HDV prevalence in Canada using a comprehensive literature review and meta-analysis. A comprehensive literature review of articles reporting HBsAg seroprevalence and anti-HDV prevalence was conducted to calculate country-specific rates and pooled prevalence of CHB and HDV using meta-analyses. Country-specific CHB and HDV rate estimates were combined with number of FB persons in Canada in 2021 from Statistics Canada to estimate total numbers of FB with CHB and HDV, respectively. These estimates were combined with estimates of Canada-born persons with CHB and HDV to yield the total number of persons with CHB and HDV. In 2021, we estimated 0.550 million (M) (95% CI 0.488-0.615) persons with CHB; 0.344 M (95% CI 0.288-0.401) were FB and 0.206 M (95% CI: 0.200-0.214) were Canada-born. The weighted average HDV prevalence among FB persons in Canada was 5.19% (17,848 [95% CI 9611-26,052] persons), among whom 50% emigrated from Asia and 31% from Africa. When combined with estimates of Canada-born persons with HDV, we estimate 35,059 (95% CI: 18,744-52,083) persons with HDV in Canada. In conclusion, we estimate 0.550 M and 35,059 persons living with CHB and HDV, respectively, in Canada in 2021.

Neutralizing antibodies to interferon alfa arising during peginterferon therapy of chronic hepatitis B in children and adults: Results from the HBRN Trials.

BACKGROUND AIMS: Pegylated interferon-α (PegIFNα) is of limited utility during immunotolerant or immune active phases of chronic hepatitis B infection but is being explored as part of new cure regimens. Low/absent levels of IFNα found in some patients receiving treatment are associated with limited/no virological responses. The study aimed to determine if sera from participants inhibit IFNα activity and/or contain therapy-induced anti-IFNα antibodies. APPROACH RESULTS: Pre-treatment, on-treatment, and post-treatment sera from 61 immunotolerant trial participants on PegIFNα/entecavir therapy and 88 immune active trial participants on PegIFNα/tenofovir therapy were screened for anti-IFNα antibodies by indirect ELISA. The neutralization capacity of antibodies was measured by preincubation of sera±recombinant human IFNα added to Huh7 cells with the measurement of interferon-stimulated gene (ISG)-induction by qPCR. Correlations between serum-induced ISG inhibition, presence, and titer of anti-IFNα antibodies and virological responses were evaluated. Preincubation of on-treatment serum from 26 immunotolerant (43%) and 13 immune active (15%) participants with recombinant-human IFNα markedly blunted ISG-induction in Huh7 cells. The degree of ISG inhibition correlated with IFNα antibody titer ( p < 0.0001; r = 0.87). On-treatment development of anti-IFNα neutralizing antibodies (nAbs) was associated with reduced quantitative HBsAg and qHBeAg declines ( p < 0.05) and inhibited IFNα bioactivity to 240 weeks after PegIFNα cessation. Children developed anti-IFNα nAbs more frequently than adults ( p = 0.004) but nAbs in children had less impact on virological responses. CONCLUSIONS: The development of anti-IFNα nAbs during PegIFNα treatment diminishes responses to antiviral therapy. Understanding how and why anti-IFNα antibodies develop may allow for the optimization of IFN-based therapy, which is critical given its renewed use in HBV-cure strategies.

Feasibility of hepatitis C elimination by screening and treatment alone in high-income countries.

BACKGROUND AND AIMS: Despite the availability of highly effective direct-acting antiviral therapy, chronic hepatitis C (CHC) continues to cause a major public health burden. In many high-income countries, treatment rates have been declining, which was exacerbated by the impact of the COVID-19 pandemic, threatening the ability to meet the World Health Organization (WHO)'s targets for eliminating HCV as a public health threat by 2030. We sought to model the impact of CHC in Canada, a resource-rich country with ongoing immigration from HCV-endemic regions; which relies exclusively on risk-based screening for case identification. APPROACH AND RESULTS: We developed an agent-based model to characterize the HCV epidemic in a high-income country with ongoing immigration. Combinations of prevention such as harm reduction, screening, and treatment strategies were considered. Model parameters were estimated from the literature and calibrated against historical HCV data. Sensitivity analyses were performed to assess uncertainty. Under the current status quo of risk-based screening, we predict the incidence of CHC-induced decompensated cirrhosis, HCC, and liver-related deaths would decrease by 79.4%, 76.1%, and 62.1%, respectively, between 2015 and 2030, but CHC incidence would only decrease by 11.1%. The results were sensitive to HCV transmission rate and an annual number of people initiating treatment. CONCLUSIONS: Current risk-based screening, and subsequent treatment, will be inadequate to achieve WHO goals. With extensive scale-up in screening, and treatment, the mortality target may be achievable, but the target for preventing new CHC cases is unlikely reachable, highlighting the importance of developing enhanced harm-reduction strategies for HCV elimination.

Evaluation of Prenatal Hepatitis C Virus Prevalence Using Universal Screening, and Linkage to Care in a Real-World Setting in Ontario.

OBJECTIVES: International infectious disease/obstetrical societies have recently recommended universal hepatitis C virus (HCV) prenatal screening and these same recommendations are forthcoming in Canada. At present, there is no formal analysis of universal HCV screening or linkage to care of pregnant people in Ontario. The objectives of our study were to determine the seroprevalence of HCV using 2 different methods to evaluate universal screening, as well as identify opportunities that may improve linkage to care. METHODS: To assess seroprevalence in a large urban area, we aimed to test 12 000 de-identified samples submitted for prenatal HIV testing in the catchment area of Toronto Public Health for HCV antibodies. Then, to assess the seroprevalence as well as the operational impact and follow-up in a real-world setting, we completed a Quality Improvement Project (QIP) for 1 year at a large tertiary care obstetrical centre in London, Ontario. RESULTS: From 2019 to 2021, 11 999 de-identified samples were screened from Toronto with a seroprevalence of 0.40 (95% CI 0.29-0.53). In London, 5771 people were screened in 2021 with a seroprevalence of 0.55% (95% CI 0.38-0.78). Taken together, those aged 26-35 years had the highest positivity; in the QIP, 9% had no documented risk factor, and 59% of individuals were not linked to the next step in HCV care. CONCLUSIONS: HCV prenatal seroprevalence in Ontario is comparable to hepatitis B virus, and ∼15-30-fold higher than HIV. Diagnosis in pregnancy is critical to facilitate referrals for treatment between pregnancies and could increase screening among children born to positive women.

Validation of case-ascertainment algorithms using health administrative data to identify people who inject drugs in Ontario, Canada.

OBJECTIVES: Health administrative data can be used to improve the health of people who inject drugs by informing public health surveillance and program planning, monitoring, and evaluation. However, methodological gaps in the use of these data persist due to challenges in accurately identifying injection drug use (IDU) at the population level. In this study, we validated case-ascertainment algorithms for identifying people who inject drugs using health administrative data in Ontario, Canada. STUDY DESIGN AND SETTING: Data from cohorts of people with recent (past 12 months) IDU, including those participating in community-based research studies or seeking drug treatment, were linked to health administrative data in Ontario from 1992 to 2020. We assessed the validity of algorithms to identify IDU over varying look-back periods (ie, all years of data [1992 onwards] or within the past 1-5 years), including inpatient and outpatient physician billing claims for drug use, emergency department (ED) visits or hospitalizations for drug use or injection-related infections, and opioid agonist treatment (OAT). RESULTS: Algorithms were validated using data from 15,241 people with recent IDU (918 in community cohorts and 14,323 seeking drug treatment). An algorithm consisting of ≥1 physician visit, ED visit, or hospitalization for drug use, or OAT record could effectively identify IDU history (91.6% sensitivity and 94.2% specificity) and recent IDU (using 3-year look back: 80.4% sensitivity, 99% specificity) among community cohorts. Algorithms were generally more sensitive among people who inject drugs seeking drug treatment. CONCLUSION: Validated algorithms using health administrative data performed well in identifying people who inject drugs. Despite their high sensitivity and specificity, the positive predictive value of these algorithms will vary depending on the underlying prevalence of IDU in the population in which they are applied.

Limited Sustained Remission After Nucleos(t)ide Analog Withdrawal: Results From a Large, Global, Multiethnic Cohort of Patients With Chronic Hepatitis B (RETRACT-B Study).

INTRODUCTION: Complete viral suppression with nucleos(t)ide analogs (NAs) has led to a profound reduction in hepatocellular carcinoma and mortality among patients with chronic hepatitis B. Finite therapy yields higher rates of functional cure; however, initial hepatitis B virus (HBV) DNA and alanine aminotransferase (ALT) elevations are almost certain after treatment interruption. We aimed to analyze off-treatment outcomes beyond 12 months after NA cessation. METHODS: Patients with well-suppressed chronic hepatitis B who were hepatitis B e antigen-negative at NA cessation and remained off treatment without hepatitis B surface antigen (HBsAg) loss at 12 months were included (n = 945). HBV DNA and ALT fluctuations were allowed within the first 12 months. We used Kaplan-Meier methods to analyze outcomes beyond 12 months. Sustained remission was defined as HBV DNA <2,000 IU/mL and ALT <2× upper limit of normal (ULN) and an ALT flare as ALT ≥5× ULN. RESULTS: Cumulative probability of sustained remission was 29.7%, virological relapse was 65.2% with a mean peak HBV DNA of 5.0 ± 1.5 log 10 IU/mL, an ALT flare was 15.6% with a median peak ALT × ULN of 8.3 (5.7-11.3), HBsAg loss was 9.9% and retreatment was 34.9% at 48 months after NA cessation. A single occurrence of virological relapse or an ALT flare within the first 12 months off-treatment were associated with significantly lower rates of sustained remission beyond 12 months. DISCUSSION: Despite allowing for HBV DNA and ALT fluctuations within the first 12 months off-treatment, most patients without HBsAg loss did not maintain a sustained response thereafter. The best candidates for NA withdrawal are patients with low HBsAg levels at NA cessation, and those without profound or recurrent virological and biochemical relapses in the first off-treatment year.

Factors associated with knowledge and awareness of Hepatitis B in individuals of Chinese descent: Results from a mass point of care testing and outreach campaign in Toronto, Canada.

Background: Migrants from hepatitis B virus (HBV) endemic regions are at high risk of having chronic infection. Despite this, HBV knowledge and awareness programming, and low-barrier screening methods such as point of care (POC) testing, among this group have yet to become routine. Methods: We conducted a mass HBV POC screening and knowledge and awareness campaign for individuals of Chinese descent in Toronto, Canada. POC screening was administered, then participants completed a knowledge questionnaire. Logistic regression identified associations between demographic factors and participants' level of HBV knowledge. Results: From 2015 to 2018, 33 outreach events resulted in 891 individuals completing testing and the knowledge questionnaire. Individuals averaged 64.4 years old. Most, 62% (N = 552), were female, and 73.6% (N = 656) have been in Canada for <30 years. The average questionnaire score was 70.7% correct, with 65.2% (N = 581) demonstrating a high level of HBV knowledge. Post-secondary education (OR: 2.19, 95% CI: 1.41, 3.39), income of $50,000 to <$75,000 (OR: 2.74, 95% CI: 1.39, 5.43), and having familial history of HBV (OR: 1.72, 95% CI: 1.06, 2.78) were associated with high knowledge. The observed prevalence of HBV was 1.5%, with 13 individuals testing positive on the POC test and confirmatory laboratory testing. Conclusions: Improving knowledge and awareness of HBV is critical to empowering people, especially migrants who experience barriers to care, to pursue vaccination, testing, and treatment. Combining knowledge outreach and POC test campaigns, enabled discussion and screening for HBV with large numbers of people, and can be tailored for optimal effectiveness for specific groups.

Test characteristics for combining non-invasive liver fibrosis staging modalities in individuals with Hepatitis C virus.

Non-invasive methods have largely replaced biopsy to identify advanced fibrosis in hepatitis C virus (HCV). Guidelines vary regarding testing strategy to balance accuracy, costs and loss to follow-up. Although individual test characteristics are well-described, data comparing the accuracy of using two tests together are limited. We calculated combined test characteristics to determine the utility of combined strategies. This study synthesizes empirical data from fibrosis staging trials and the literature to estimate test characteristics for Fibrosis-4 (FIB4), APRI or a commercial serum panel (FibroSure®), followed by transient elastography (TE) or FibroSure®. We simulated two testing strategies: (1) second test only for those with intermediate first test results (staged approach), and (2) second test for all. We summarized empiric data with multinomial distributions and used this to estimate test characteristics of each strategy on a simulated population of 10,000 individuals with 4.2% cirrhosis prevalence. Negative predictive value (NPV) for cirrhosis from a single test ranged from 98.2% (95% CB 97.6-98.8%) for FIB-4 to 99.4% (95% CB 99.0-99.8%) for TE. Using a staged approach with TE second, sensitivity for cirrhosis rose to 93.3-96.9%, NPV to 99.7-99.8%, while PPV dropped to <32%. Using TE as a second test for all minimally changed estimated test characteristics compared with the staged approach. Combining two non-invasive fibrosis tests barely improves NPV and decreases or does not change PPV compared with a single test, challenging the utility of serial testing modalities. These calculated combined test characteristics can inform best methods to identify advanced fibrosis in various populations.

Maternal neighbourhood-level social determinants of health and their association with paediatric hepatitis C screening among children exposed to hepatitis C in pregnancy.

BACKGROUND: Current guidelines recommend HCV screening by 18 months of age for those exposed to HCV in utero; yet, screening occurs in the minority of children. OBJECTIVES: To evaluate the association between maternal neighbourhood-level social determinants of health (SDOH) and paediatric HCV screening in the general population in a publicly funded healthcare system in Canada. METHODS: Retrospective cohort study using administrative healthcare data held at ICES. Children born to individuals positive for HCV RNA in pregnancy from 2000 to 2016 were identified and followed for 2 years. Major SDOH were identified, and the primary outcome was HCV screening in exposed children (HCV antibody and/or RNA). Associations between SDOH and HCV screening were determined using multivariate Poisson regression models adjusting for confounding. RESULTS: A total of 1780 children born to persons with +HCV RNA were identified, and 29% (n = 516) were screened for HCV by age two. Most mothers resided in the lowest income quintile (42%), and most vulnerable quintiles for material deprivation (41%), housing instability (38%) and ethnic diversity (26%) with 11% living in rural locations. After adjustment for confounding, maternal rural residence (risk ratio [RR] 0.82, 95% confidence interval [CI] 0.62, 1.07) and living in the highest dependency quintile (RR 0.83, 95% CI 0.65, 1.07) were the SDOH most associated with paediatric HCV screening. Younger maternal age (RR 0.98 per 1-year increase, 95% CI 0.97, 0.99), HIV co-infection (RR 1.69, 95% CI 1.16, 2.48) and GI specialist involvement (RR 1.18, 95% CI 1.00, 1.39) were associated with higher probabilities of screening. CONCLUSIONS: Among children exposed to HCV during pregnancy, rural residences and living in highly dependent neighbourhoods showed a potential association with a lower probability of HCV screening by the age of 2. Future work evaluating barriers to paediatric HCV screening among rural residing and dependent residents is needed to enhance the screening.

Characterising the effectiveness of social determinants of health-focused hepatitis B interventions: a systematic review.

Social determinants of health are important in designing effective interventions for hepatitis B virus (HBV) infection. This systematic review characterises equity-oriented, social determinants of health-focused HBV interventions, and describes their effectiveness in terms of the prevention, care, or treatment of HBV in high-income countries. We searched electronic databases for central concepts of 'HBV', 'equity', 'social determinants of health', 'intervention', and 'Organization for Economic Co-operation and Development (OECD) countries'. Screening and data abstraction were conducted independently by two reviewers. Data were abstracted from 66 studies; articles with a comparative study design (n=36) were included in the narrative synthesis, highlighting social determinants of health domains of interventions, HBV-relevant health outcomes, and extra-health social determinants of health effects (ie, those effects that extend beyond health outcomes). Synthesis aligned with six emergent themes corresponding to HBV prevention and care: knowledge and education, diagnosis and screening, immunisation, care initiation, engagement with clinical care and treatment, and upstream prevention. Studies presented a heterogeneous array of HBV-relevant health outcomes. Most interventions were tailored for social determinants of health domains of race, ethnicity, culture, and language; drug use; and socioeconomic status. Across the themes, at least two-thirds of interventions showed comparative effectiveness for addressing HBV. Extra-health social determinants of health outcomes were observed for two studies. Considerable diversity in population-level approaches was observed regarding intervention goals and effectiveness; most interventions were effective at enhancing the prevention, care, or treatment of HBV.

Addition of PEG-interferon to long-term nucleos(t)ide analogue therapy enhances HBsAg decline and clearance in HBeAg-negative chronic hepatitis B: Multicentre Randomized Trial (PAS Study).

We studied whether 48 weeks of PEG-IFN alfa-2a add-on increases HBsAg-decline and clearance in HBeAg-negative patients on long-term nucleo(s)tide analogue (NA) therapy. In this investigator-initiated, randomized, controlled trial conducted in Europe and Canada, HBeAg-negative patients treated with NA > 12 months, with HBVDNA < 200 IU/mL, were enrolled. Patients were randomized 2:1 to 48 weeks of PEG-IFN alfa-2a add-on (180 µg per week) or continued NA-monotherapy with subsequent follow-up to Week 72. Endpoints were HBsAg decline (≥1 log10 IU/mL) and HBsAg clearance at Week 48. Of the 86 patients in the modified-intention-to-treat analysis, 58 patients received PEG-IFN add-on, and 28 continued NA monotherapy. At Week 48, 16(28%) patients achieved HBsAg decline ≥1 log10 in the add-on arm versus none on NA-monotherapy (p < .001), and HBsAg clearance was observed in 6 (10%) PEG-IFN add-on patients versus 0% NA-monotherapy (p = .01). HBVRNA was only detected in 2% after PEG-IFN treatment versus 19% in NA-monotherapy (p = .002) at Week 48. PEG-IFN add-on therapy was well tolerated in majority of patients. Low baseline HBsAg levels (<10 IU/mL) identified patients most likely to achieve HBsAg loss with PEG-IFN add-on, whereas an HBsAg level > 200 IU/mL at on-treatment Week 12 was highly predictive of non-response (NPV = 100%). Addition of PEG-IFN to long-term NA enhanced HBsAg decline and increased the chance of HBsAg clearance in HBeAg-negative patients on long-term NA. On-treatment HBsAg levels >200 IU/mL identify patients unlikely to benefit from PEG-IFN add-on and could be used as a potential stopping-rule for PEG-IFN therapy. Our findings support further exploration of immune modulation add-on to antiviral therapy, preferably using response-guided strategies, to increase functional cure rates in patients with CHB.