Journal of Clinical and Translational Hepatology 2022 vol. 10(6) | 1068–1076 DOI: 10.14218/JCTH.2022.00068 Original Article ACE Score Identifies HBeAg-negative Inactive Carriers at a Single-point Evaluation, Regardless of HBV Genotype Luisa Roade1,2,3# , Mar Riveiro-Barciela1,2,3#* , Adriana Palom2,4, Francisco Rodríguez-Frías3,4,5 , Marta Bes4,6,7 , Ariadna Rando3,4,5 , María Teresa Salcedo8 , Rosario Casillas4,5 , Elena Vargas-Accarino3,6, David Tabernero3,4,5 , Silvia Sauleda4,6,7, Rafael Esteban1,2,3 and María Buti1,2,3 1LiverUnit, Internal Medicine Department, Hospital Universitari Vall d’Hebron, Vall d’Hebron Barcelona Hospital Campus, Barcelona, Spain; 2Universitat Autònoma de Barcelona (UAB), Department of Medicine, Barcelona, Spain; 3Universitat Au- tònoma de Barcelona (UAB), Department of Biochemistry and Molecular Biology, Barcelona, Spain; 4Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain; 5Liver Pathology Lab, Biochemistry and Microbiology Departments (Clinical Laboratories), Hospital Universitari Vall d’Hebron, Vall d’Hebron Barcelona Hospital Campus, Barcelona, Spain; 6Vall d’Hebron Institut de Recerca (VHIR), Liver Diseases Group, Barcelona, Spain; 7Transfusion Safety Laboratory, Banc de Sang i Teixits, Servei Català de la Salut, Barcelona, Spain; 8De- partment of Pathology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona, Spain Received: 9 February 2022 | Revised: 25 March 2022 | Accepted: 29 March 2022 | Published: 24 April 2022 Abstract yielded an area under the receiver operating characteris- tics (AUROC) of 0.925 (95% CI: 0.880–0.970, p<0.001) for identification of ICs. The AUROC for genotype D was 0.95, Background and Aims: Hepatitis B virus (HBV) biomark- 0.96 for A, 0.90 for E, and 0.88 for H/F. An ACE score of <1 ers have been used for a better categorization of patients, had a positive predictive value of 99.5%, and a score ≤12 even though the lack of simple algorithms and the impact of points had a diagnostic accuracy of 93.8%. Conclusions: genotypes limit their application. Our aim was to assess the Low baseline HBV DNA, HBcrAg, and liver stiffness were in- usefulness of noninvasive markers for the identification of dependently associated with the IC phase. A score including HBV inactive carriers (ICs) in a single-point evaluation and those variables identified ICs at a single-point evaluation, to design a predictive model for their identification. Meth- and might be applied to implement less intensive follow-up ods: This retrospective-prospective study included 343 strategies. consecutive HBeAg-negative individuals. Clinical, analytical, and virological data were collected, and a liver biopsy was Citation of this article: Roade L, Riveiro-Barciela M, Palom performed if needed. Subjects were classified at the end of A, Rodríguez-Frías F, Bes M, Rando A, et al. ACE Score Identi- follow-up as ICs, chronic hepatitis B and gray zone.A pre- fies HBeAg-negative Inactive Carriers at a Single-point Eval- dictive model was constructed, and validated by 1000-boot- uation, Regardless of HBV Genotype. J Clin Transl Hepatol strap samples. Results: After 39 months of follow-up, 298 2022;10(6):1068–1076. doi: 10.14218/JCTH.2022.00068. subjects were ICs, 36 were chronic hepatitis B CHB, and nine were gray zone. Eighty-nine (25.9%) individuals re- quired a liver biopsy. Baseline HBV DNA hazard ratio (HR) 6.0, p<0.001), HBV core-related antigen (HBcrAg) (HR 6.5, Introduction p<0.001), and elastography (HR 4.6, p<0.001) were inde- pendently associated with the IC stage. The ACE score (HBV DNA, HBcrAg, elastography), obtained by bootstrapping, Chronic hepatitis B infection affects 296 million people worldwide according to the World Health Organization.1 The course of chronic hepatitis B infection is described in Keywords: Hepatitis B virus; Inactive carrier; Liver stiffness; HBV DNA; Quan- different phases as a result of complex and dynamic in- titative HBsAg; Core-related antigen. teractions between the host immunity and the viral par- Abbreviations: ALT, alanine aminotransferase; APRI, AST-to-platelet ratio in- ticles.2 Hepatitis B e antigen (HBeAg)-negative stage dex; AST, aspartate aminotransferase; AUROC, area under the receiver operat- ing characteristic; CHB, chronic hepatitis B; C-index, concordance index; FIB-4, represents the vast majority of chronic HBV infections in fibrosis-4 index; GGT, gamma glutamyl transferase; GZ, gray zone; HBcrAg, Western countries.3,4 The risk of cirrhosis, liver cancer, de- hepatitis B core-related antigen; HBeAg, hepatitis B e antigen; HBsAg, hepa- compensation, and liver-related death dramatically varies titis B surface antigen; HBV, hepatitis B virus; HCV, hepatitis C virus; HR, haz- throughout the HBeAg-negative infection phases, which ard ratio; IC, inactive carriers; IQR, interquartile range; qHBsAg, quantitative hepatitis B surface antigen; LLD, lower limit of detection; LLQ, lower limit of are not always consecutive and whose duration may differ quantification; LSM, liver stiffness measurement; ULN, upper limit of normal. among individuals. Assessment of HBV infection by alanine #Contributed equally to this work. aminotransferase (ALT) and HBV viral load is not always *Correspondence to: Mar Riveiro-Barciela, Passeig de la Vall d’Hebron 119- enough to correctly categorize the disease stage because 129, 08035, Barcelona, Liver Unit, Department of Internal Medicine, Hospital Universitari Vall d’Hebron, Barcelona, Spain. ORCID: https://orcid.org/0000- of frequent fluctuations of both markers. Noninvasive tools 0001-9309-2052. Tel: +93-4893030-6559, Fax: +93-4894032, E-Mail: mar. such as serum markers and liver stiffness have been tested riveiro@gmail.com as complementary information to determine HBV infection Copyright: © 2022 The Author(s). This article has been published under the terms of Creative Commons Attribution-Noncommercial 4.0 International License (CC BY-NC 4.0), which permits noncommercial unrestricted use, distribution, and reproduction in any medium, provided that the following statement is provided. “This article has been published in Journal of Clinical and Translational Hepatology at https://doi.org/10.14218/JCTH.2022.00068 and can also be viewed on the Journal’s website at http://www.jcthnet.com”. Roade L. et al: ACE score identifies HBV inactive carriers phase.5,6 However, the accuracy of these markers is not groups: well standardized and some of them, such as quantification • Normal ALT and HBV DNA <2,000 IU/mL; of hepatitis B surface antigen (HBsAg), have been shown • ALT > two-fold upper limit of normal (ULN) and HBV DNA to be deeply influenced by the HBV genotype.7,8 These fac- > 20,000 IU/mL; tors make difficult an accurate assessment and prediction • Subjects who did not fulfill any of the above conditions. of the long-term outcome of HBeAg-negative subjects by a The ALT ULN was defined by local reference laboratory single evaluation. As a result, a follow-up of three medical values of 35 IU/mL for women and 50 IU/mL for men. Sub- visits within the first year is currently recommended to de- jects were followed by different hepatologists according to fine the phase of infection.9 Although that approach reflects the same protocol. Following the guideline recommenda- the changing character of the infection, one-point assess- tions, liver biopsies were performed in subjects with HBV ments should be developed in real-life cohorts to facilitate DNA persistently above 2,000 IU/mL and normal ALT or ALT decentralized models of care and simplified algorithms. <2-fold the ULN during follow-up.13 Liver specimens were One-point assessments are especially important because read by the same pathologist. Significant fibrosis was es- of the endemicity of HBV infection in vulnerable populations tablished in fibrosis stage ≥3 according to the Ishak score.14 and in low and middle-resource regions such as Sub-Saha- Subjects with at least one follow-up visit were reclassified, ran Africa, the Western Pacific, and Southeast Asia, which taking serum ALT, HBV DNA levels into consideration, and account for more than 80% of infections worldwide.1,10 A liver fibrosis stage by histological sample when needed, fol- proper classification is essential to identify not only indi- lowing European Association for the Study of the Liver 2017 viduals at increased risk of disease progression, but also Clinical Practice Guidelines.13,14 those in an inactive phase of infection who would benefit Chronic HBeAg-negative infection ICs was persistently from less intensive management strategies. The aim of this normal ALT and HBV DNA of <2,000 IU/mL or HBV DNA study was to assess the usefulness of noninvasive markers between 2,000–20,000 IU/mL in the absence of significant to identify subjects with HBeAg-negative chronic infection, fibrosis in liver biopsy. namely former inactive carriers (ICs), and to develop a pre- Chronic HBeAg-negative hepatitis (CHB) was elevated dictive model for early identification of these subjects in a ALT and HBV DNA >2,000 IU/mL, and/or significant fibrosis. single-point evaluation. Gray zone (GZ) was persistently normal ALT and HBV DNA >20,000 IU/mL in the absence of significant fibrosis. Liver cirrhosis was diagnosed by either imaging findings Methods (irregular liver surface and direct/indirect signs of portal hy- pertension) or liver histology with an Ishak fibrosis score A retrospective-prospective cohort study was performed of 5–6. Subjects with liver ultrasound and/or histological in a university hospital in Barcelona, Spain. Subjects aged signs of liver cirrhosis were considered as having CHB infec- over 16 years of age with HBsAg documented for at least 6 tion, regardless of their ALT and HBV DNA levels. Supple- months who attended the outpatient department between mentary Figure 1 summarizes the study design. Participant July 2013 and December 2019 were included. Subjects who data were anonymized and informed consent was waived were lost follow-up after the first medical visit were ex- because of the study design. The preparation of this manu- cluded. Subjects who tested positive for HBeAg at the first script was performed following STROBE guidelines. visit and/or with hepatitis C virus (HCV), hepatitis D virus, and human immunodeficiency virus coinfection defined as positivity for both serology and viral load, history of alcohol Statistical analysis abuse and/or evidence of autoimmune liver disease were also excluded. Demographic, clinical, and anthropometric Quantitative variables with a normal distribution were re- variables were collected in the first medical visit. Labora- ported as means and standard deviation. Non-normally tory data and noninvasive markers of liver fibrosis were re- distributed quantitative variables were reported as me- corded at baseline, 6 months, and yearly. Hepatitis B infec- dians and interquartile range (IQR). Comparisons were tion parameters included quantitative HBsAg (qHBsAg). The performed with Student t-test and Mann-Whitney U-test. lower limit of quantification (LLQ) was 0.05 IU/mL, HBeAg, Categorical variables were described as absolute and rela- and anti-HBe, all tested by commercially available electro- tive frequencies (percentages, %) and compared with chi- chemiluminescence immunoassays (COBAS 8000; Roche square or Fisher’s exact tests in case of relative frequen- Diagnostics, Rotkreuz, Switzerland). Serum HBV DNA was cies below 5%. Baseline variables that had a clinically and measured with a commercial PCR assay that had an LLQ statistically significant association to the outcome in uni- of 20 IU/mL and lower limit of detection (LLD) of 10 IU/ variate analysis (Mantel–Cox test) were selected for the in- mL (COBAS 6800; Roche Diagnostics, Manheim, Germany). itial models (p<0.10). The final models were obtained by a HBV core-related antigen (HBcrAg) was measured with a stepwise forward method based on model likelihood ratios chemiluminescent enzyme immunoassay (CLIA, Lumipulse (Cox regression). The same significance level (p<0.05) G HBcrAg assay; Fujirebio, Gent, Belgium) that had an LLD was set for including and discarding variables. Quantitative of 3 logU/mL. For HBV genotyping, HBV DNA was first en- variables included in the models were categorized by clini- riched by ultracentrifugation of 9.6 mL of serum and Sanger cally significant cutoffs in order to increase the power. The sequencing was carried out after amplification of two dif- model obtained was calibrated by a 1000-bootstrapping ferent viral regions, PreC/Core (nucleotides 1,774–2,389, analysis to minimize overfit bias.15 A weighted semiquanti- 615 bp) and PreS/Surface (nucleotides 2,828–176,561 bp), tative score was constructed based on the final model. The as previously described.11 Genotypes H and F were com- score for each variable reflected the risk coefficient ob- bined because of their phylogenetic proximity and similar tained after the bootstrapping analysis. The discrimination geographic distribution.11,12 Noninvasive fibrosis mark- performance of the obtained predictive models was evalu- ers included liver stiffness measurement-LSM (FibroScan) ated with receiver operating characteristic (ROC) curve and the serum biomarkers Fibrosis-4 index (FIB-4) and the analysis and the concordance index (C-index). The cutoff aspartate aminotransferase (AST)-to-platelet ratio index values were selected considering the highest Youden’s in- (APRI). dex, and expressed as sensitivity, specificity, and predic- According to the one-time assessment at the first study tive value. The results were considered statistically signifi- visit, HBeAg-negative subjects were preclassified in three cant when the p-value was <0.05. The statistical analysis Journal of Clinical and Translational Hepatology 2022 vol. 10(6) | 1068–1076 1069 Roade L. et al: ACE score identifies HBV inactive carriers Table 1. Baseline characteristics in the overall cohort and the final classification Inactive carri- Gray zone Chronic hepa- Overall (n=343) p-value ers (n=298) (n=9) titis B (n=36) Male gender 203 (59.2%) 173 (58.1%) 4 (44.4%) 26 (72.2%) 0.174 Age (years) 44.5±14.6 44.9±14.4 38.2±17.5 43.4±15.1 0.360 Ethnicity   Caucasian 217 (63.3%) 194 (65.1%) 4 (44.4%) 19 (52.8%) 0.060   Black 79 (23.0%) 63 (21.1%) 5 (55.6%) 11 (30.6%) 0.060   Asian 23 (6.7%) 18 (6.0%) 0 (0%) 5 (13.9%) 0.060   Hispanic 24 (7.0%) 23 (7.7%) 0 (0%) 1 (2.8%) 0.060 Comorbidities   Obesity 60 (17.5%) 56 (23.0%) 1 (11.1%) 3 (10.7%) 0.240   Dyslipidemia 53 (15.5%) 51 (17.5% 1 (11.1%) 1 (2.8%) 0.075   Arterial hypertension 53 (15.5%) 48 (16.1%) 1 (11.1%) 4 (11.1%) 0.685   Diabetes mellitus 14 (4.1%) 11 (3.7%) – 3 (8.3%) 0.339 Liver cirrhosis 8 (2.3%) – – 8 (22.2%) <0.001 Platelets (×109/mm3) 225±58,000 228±57 245±58 198±58 0.009 ALT (IU/L) 28±21 25±11 29±15 56±46 <0.001 HBV DNA (log IU/mL) 2.8±1.2 2.6±1.0 3.4±0.9 4.4±1.3 <0.001 qHBsAg (log IU/mL) 3.1±1.1 3.0±1.1 3.9±0.6 3.7±0.6 <0.001 qHBsAg >1,000 IU/mL1 205 (60.5%) 168 (56.9%) 8 (88.9%) 29 (82.9%) 0.003 HBcrAg (log U/mL)2   <3 logU/mL 274 (79.9%) 258 (91.5%) 4 (44.4%) 12 (37.5%) <0.001   3–4 logU/mL 38 (11.1%) 23 (8.2%) 4 (44.4%) 11(34.4%) <0.001   4–5 logU/mL 8 (2.3%) 1(0.4%) 1 (11.1%) 6 (18.8%) <0.001   >5 logU/mL 3 (0.9%) – – 3 (9.4%) <0.001 Genotype3   D 102 (40.8%) 93 (43.1%) 2 (22.2%) 7 (28.0%) 0.209   A 68 (27.2%) 61 (28.2%) 2 (22.2%) 5 (20.0%) 0.209   E 40 (16.0%) 31 (14.4%) 3 (33.3%) 6 (24.0%) 0.209   F/H 26 (10.4%) 20 (9.3%) 2 (22.2%) 4 (16.0%) 0.209   B/C 10 (4.0%) 7 (3.2%) – 3 (12.0%) 0.209   Mixed 4 (1.6%) 4 (1.9%) – – 0.209 Elastography (kPa) 5.6±2.3 5.2±1.7 6.9±1.5 8.2±4.3 <0.001 FIB-4 0.5±0.4 0.5±0.4 0.4±0.2 0.5±0.7 0.617 APRI 0.5±0.4 0.4±0.2 0.5±0.3 0.9±0.9 <0.001 Categorical variables are n (%), quantitative variables are means ± SD. 1qHBsAg was available in 339 subjects of the overall cohort (295 inactive carriers, nine gray zone, 35 chronic hepatitis); 2HBcrAg was available in 323 subjects; 3HBV-genotype was available in 250 subjects of the overall cohort (216 inactive carriers, nine gray zone, 25 chronic hepatitis B). ALT, alanine aminotransferase; APRI, ALT to platelet ratio index; FIB-4, fibrosis-4 index; HBV, hepatitis B virus; HBcrAg, hepatitis B core- related antigen; qHBsAg, quantitative hepatitis B surface antigen. were performed with IBM SPSS, version 26.0 (IBM Corp, cluded (Table 1). Most were male (59.2%), Caucasian Armonk, NY, USA). (63.3%), and the mean age was 45 years. Black individuals represented a considerable percentage of the overall cohort (23.0%), most of them coming from Western African coun- Results tries, and 179 subjects (52.1%) were immigrants from non- Western European regions. The HBV genotype was deter- Baseline characteristics mined in 250 individuals; D and A were the most prevalent, followed by E and H/F. Figure 1 summarizes the country of origin and the most prevalent HBV genotypes among im- Three hundred forty-three consecutive subjects were in- migrants. At the first visit, most subjects (68.8%) had a 1070 Journal of Clinical and Translational Hepatology 2022 vol. 10(6) | 1068–1076 Roade L. et al: ACE score identifies HBV inactive carriers Fig. 1. Country of origin and most prevalent HBV genotype of immigrants in the overall cohort (by relative frequency). HBV, hepatitis B virus. normal ALT and HBV DNA <2,000 IU/mL, five (1.5%) had liver samples (p=0.05, Supplementary Fig. 2).5,16 FIB-4 an ALT >2× the ULN and HBV DNA >20,000 IU/mL, and 102 values did not differ in the presence of significant fibrosis, (29.7%) did not fulfill the conditions for any of the above but those with ≥F3 had higher APRI levels (p=0.048). Mul- categories (Fig. 1). tivariate analysis found that LSM categories (HR 3.37, 95% CI: 1.08–10.49, p=0.037) and baseline HBcrAg (HR 3.615, 95% CI: 1.41–9.25, p=0.007) were independently associ- Clinical follow-up and liver biopsy ated with significant fibrosis. Two hundred fifty-four (74.1%) subjects were classified by Classification of the HBV phase during follow-up a noninvasive approach, and liver biopsies were needed in 89 (25.9%) for proper classification of the HBV phase. Twenty-eight (12.1%) of two hundred thirty-one subjects Subjects were classified after a mean follow-up of 39.0 presented with baseline normal ALT and an HBV DNA months. Figure 2 shows the changes from the initial to the <2,000 IU/mL, and sixty-one (61.0%) of 100 were initially final classification. At follow-up, 298 subjects were consid- considered as GZ. The median time from the first visit to ered ICs, 36 patients were found to have CHB, and nine liver biopsy was 8.4 months (IQR 2.2–15.3). Nine out of subjects remained in in the GZ, all had viral loads persis- 89 (10.1%) subjects presented significant fibrosis (≥F3), tently over 20,000 UI/mL, normal ALT, and non-significant whereas 73 (82.0%) presented Ishak score below F1. Table fibrosis on liver biopsy. Of the 236 subjects with normal ALT 2 summarizes baseline features by fibrosis stage, in sub- and HBV DNA <2,000 IU/mL, 230 (97.5%) were consid- jects who underwent invasive management. Significant fi- ered ICs during follow-up and six (2.5%) were regarded as brosis was significantly more frequent in males, whereas no CHB after the histological assessment. Of 102 subjects in differences were found in baseline ALT, qHBsAg and HBV- the GZ group after the one-point assessment at baseline, DNA levels. However, significantly higher baseline HBcrAg 68 (66.7%) were finally classified as ICs and 25 (24.5%) levels were observed in subjects with significant fibrosis as CHB. All five subjects with initial ALT levels >2 times (p<0.001). LSM tended to be lower among subjects without the ULN and HBV DNA >20,000 IU/mL remained a the CHB significant fibrosis (p=0.085). However, when categorizing stage. liver stiffness measurements with a double cutoff system Baseline features by final classification are summarized in (i.e. below 6.5 kPa, between 6.5 and 9 kPa and above 9 Table 1. Ethnic distribution tended to differ (p=0.060), with kPa), a significant association was found between elastog- a higher proportion of black individuals in the GZ (55.6%). raphy categories and the presence of significant fibrosis in The prevalence of transmission pathways, toxic habits, Journal of Clinical and Translational Hepatology 2022 vol. 10(6) | 1068–1076 1071 Roade L. et al: ACE score identifies HBV inactive carriers Table 2. Baseline features of individuals who required a liver biopsy during follow-up for proper classification of HBV infection Non-significant fibro- Significant fibro- Univariate analysis sis (1,000 IU/mL 63 (79.7%) 8 (88.9%) 0.447 HBcrAg (log U/mL) <3 logU/mL 64 (81.0%) 5 (62.5%) 3–4 logU/mL 11 (13.9%) – 4–5 logU/mL 4 (5.1%) – >5 logU/mL – 3 (37.5%) HBV DNA (log IU/mL) 3.6±0.9 4.2±1.3 0.079 Genotype   D 23 (37.1%) 2 (40%) 0.139   A 16 (25.8%) 0 (0%) 0.139   B/C 3 (4.8%) 0 (0%) 0.139   F/H 10 (16.1%) 0 (0%) 0.139   E 10 (16.1%) 3 (60%) 0.139 Elastography (kPa) 6.1±2.3 7.5±1.9 0.085 FIB-4 0.4±0.2 0.3±0.1 0.092 APRI 0.4±0.2 0.6±0.2 0.048 Categorical variables are n (%), quantitative variables are means±standard deviation. ALT, alanine aminotransferase; APRI, ALT to platelet ratio index; FIB-4, fibrosis-4 index; HBcrAg, hepatitis B core-related antigen; qHBsAg, quantitative hepatitis B surface antigen; HBV, hepatitis B virus. and comorbidities (i.e. diabetes mellitus, arterial hyperten- (95% CI: 2.3–9.0), p<0.001, for LSM; and HR 6.5 (95% sion, and dyslipidemia) was similar in all three phases of CI: 2.7–15.7, p<0.001) for HBcrAg. An ROC model based infection. Significant differences were found in baseline ALT on these coefficients yielded an AUROC of 0.925 (95% CI: (p<0.001) HBV DNA (p<0.001), qHBsAg (p<0.001) and 0.880–0.970, p<0.001) for the identification of ICs (Fig. 3). HBcrAg levels (p<0.001), as well as LSM and APRI at first The model was validated in most prevalent HBV genotypes visit; no differences were found in FIB-4 score (p=0.617). and had an area under ROC (AUROC) of 0.95 for genotype Genotype distribution was not significantly different in the D, 0.96 for A, 0.90 for E and 0.88 for H/F (Table 4). An in- three phases of infection (p=0.209). dividual-score system, the ACE score, was constructed from simplified coefficients in the bootstrapping analysis (Table 5) and included HBV DNA, HBCore-related antigen, and Markers for identification of HBV ICs liver elastography). The ACE score had the highest positive predictive value for identification of ICs for patients with Baseline markers associated with IC in the univariate analy- punctuations <1 point, and 12 points was the cutoff with sis were lower levels of ALT, AST, gamma glutamyl trans- the greatest diagnostic accuracy (93.8%). The accuracy of ferase (GGT), higher platelet count, lower HBcrAg, HBV the different cutoffs of the ACE score are summarized in DNA, and LSM. Independent association was confirmed in Table 6. the multivariate analysis between the IC group and lower HBV DNA, HBcrAg levels and LSM (Table 3). Risk coefficients Discussion similar to those obtained by multivariate analysis were ob- tained by 1000 bootstrapping samples, HR 6.0 (95% CI: 3.0–12.0), p<0.001 for categorized HBV DNA, HR 4.6 Herein we identified baseline LSM, HBV DNA, and HBcrAg 1072 Journal of Clinical and Translational Hepatology 2022 vol. 10(6) | 1068–1076 Roade L. et al: ACE score identifies HBV inactive carriers Fig. 2. HBV infection phase evolution and liver biopsy performance during follow-up. *Liver biopsy indication was established by normal ALT plus HBV DNA persistently above 2,000 IU/mL, or ALT <2-fold ULN plus viral load above 2,000 IU/mL during follow-up. **Final classification was carried out according to European Association for the Study of the Liver 2017 Clinical Practice Guidelines.16 Chronic HBeAg-negative infection-inactive carriers had persistently normal ALT plus HBV DNA <2,000 IU/mL or HBV DNA between 2,000 and 20,000 IU/mL in the absence of significant fibrosis in liver biopsy. Chronic HBeAg negative hepatitis required elevated ALT and HBV DNA >2,000 IU/mL and/or significant fibrosis at liver biopsy. Gray zone required persistently normal ALT and HBV DNA >20,000 IU/mL in the absence of fibrosis in liver biopsy. ALT, alanine aminotransferase; HBV, hepatitis B virus; ULN, upper limit of normal. Table 3. Univariate and multivariate COX proportional regression analysis of baseline factors associated with the follow-up classification as chronic infection-inactive carriers Univariate analysis Multivariate analysis n=336 p-value HR (95%CI) p-value Male sex 0.176 – – Caucasian ethnicity 0.051 – 0.378 Age 0.288 – – ALT (IU/L) <0.001 – 0.179 AST (IU/L) <0.001 – 0.323 GGT (IU/L) <0.001 – 0.269 Platelets (cells/mL) 0.029 – 0.996 qHBsAg (log IU/L)1 <0.001 – 0.111 HBV DNA (by category)2 <0.001 5.9 (2.9–11.9) <0.001 HBcrAg (log U/mL)3 <0.001 6.3 (2.6–15.1) <0.001 Elastography (by category)2 <0.001 4.0 (2.0–7.9) <0.001 1qHBsAg was available in 339 subjects of the overall cohort; 2HBcrAg was available in 323 subjects; 3Categories were introduced according to their most commonly used cutoffs: HBV DNA <2.000 IU/mL, 2,000–20,000 IU/mL, >20.000 IU/mL; liver stiffness: <6.5 kPa, 6.5–9 kPa, >9 kPa. ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, gamma glutamyl transferase; HBcrAg, hepatitis B core-related antigen; qHBsAg, quantitative hepatitis B surface antigen; HBV, hepatitis B virus. Journal of Clinical and Translational Hepatology 2022 vol. 10(6) | 1068–1076 1073 Roade L. et al: ACE score identifies HBV inactive carriers Table 5. Score system based on the novel model Score HBV DNA (IU/L)   <2,000 IU/L 0   2,000–20,000 IU/L 6   >20,000 IU/L 12 Liver stiffness (kPa)   <6.5 kPa 0   6.5–9.0 kPa 5   >9.0 kPa 10 HBcrAg (logU/mL) ≤3 logU/mL 0 3–4 logU/mL 6 4–5 logU/mL 12 Fig. 3. Area under the receiver operating characteristic (AUROC) of the >5 logU/mL 18 model for identification of HBeAg-negative chronic infection-inactive carriers subjects. HBcrAg, hepatitis B core-related antigen. values as independent predictors for the identification of cation was limited to HBV-genotype B and C.23 The same HBV ICs. We performed a retrospective-prospective real- cutoff was proposed in an Italian cohort of subjects in- life cohort study, with the development of a scoring system fected with genotype D.24 However, it is difficult to general- (ACE score) that combined the baseline variables. The score ize the results for the overall HBV population, as qHBsAg had a high specificity and positive predictive value, which has been shown to significantly vary among different HBV implies a trustworthy identification of IC subjects with a low genotypes, which limits its application in genotype-diverse risk of disease progression and good performance regard- cohorts.7,11 HBcrAg was later postulated as a surrogate less of the HBV genotype. marker of intrahepatic cccDNA.25 Significant variation in Classically, efforts have been made to identify HBV-in- HBcrAg levels was detected throughout the different HBV fected subjects at increased risk of developing liver-related infection phases,11 with the lowest titers detected in the complications,17 as antiviral treatment with high-barrier ICs.26 Interestingly, in another study including HBV geno- nucleos(t)ides analogues is effective, affordable, and has a types E and H/F, HBcrAg <3 logU/mL combined with HBV great impact on disease progression and survival.18,19 How- DNA <2,000 IU/mL had a diagnostic accuracy of 85% for ever, a more recent approach focuses not only on identify- identification of ICs regardless of HBV genotype.11 Recent- ing subjects at increased risk, but also those in the inactive ly, a multicenter European study including 1,582 HBeAg- phase of the disease who would benefit from less intensive negative subjects, HBcrAg <3 logU/mL had an AUROC of follow-up and management.20,21 Individuals in this phase 0.968 for identification of ICs.27 LSM in HBV infection is not have benign outcomes with morbidity and mortality similar as well standardized as in HCV. Double cutoff systems have to those of the general population.22 An easy and accurate been proposed to improve performance, although there is identification of ICs in a single assessment would facilitate no consensus among the different guidelines, which hin- decentralization strategies for HBV follow-up. ders its application in daily practice.28,29 An Italian study Many recent studies have approached the identification reported a combination of HBsAg, LSM, and HBV DNA with of ICs using HBV biomarkers and their combination. HB- 100% specificity, but no data regarding HBV genotype sAg titles have been proposed as a useful tool to identify were available.30 In fact, to the best of our knowledge, no IC because of their correlation with HBV DNA levels. An algorithms including LSM have been developed and vali- algorithm based on a single-point determination of qHB- dated in all HBV genotypes. sAg, ALT, and HBV DNA was described in a large Taiwanese On the other hand, the inaccuracy of noninvasive fi- cohort of HBeAg-negative subjects with HBV DNA 2,000 brosis markers in the GZ usually leads to the necessity IU/mL (ERADICATE-B cohort). The algorithm proposed the of performing a liver biopsy, which is considered the gold use of qHBsAg <1,000 IU/mL for the identification of sub- standard for fibrosis assessment. In our study roughly jects at minimal risk of disease progression, but its appli- 25% of patients needed a liver biopsy, 10% of whom had significant fibrosis that was independently associated with Table 4. Area under the receiver operating characteristic (AUROC) of higher HBcrAg levels and LSM. The relatively high percent- the model for inactive carrier identification by HBV genotype age of patients who required a liver biopsy for classifica- Genotype n AUROC 95% CI p-value tion of HBV phase, reinforces the need to optimize the use of noninvasive strategies and to develop pan-genotypic scores. Our cohort included mainly middle-aged subjects in the D 98 0.955 0.91–1.0 <0.001 IC phase, which is consistent with the current epidemiologi- A 67 0.963 0.89–1.0 <0.001 cal profile of the HBV infection in Western countries.31 On the other hand, almost half of our cohort were non-Europe- E 37 0.903 0.79–1.0 <0.001 an migrants, which probably explains the genotype distribu- H/F 26 0.883 0.75–1.0 0.005 tion compared with other European cohorts.26,32 The high proportion of migrants in our cohort should be a reminder AUROC, area under the receiver operating characteristic; CI, confidence interval. of the need of the integration of viral hepatitis management 1074 Journal of Clinical and Translational Hepatology 2022 vol. 10(6) | 1068–1076 Roade L. et al: ACE score identifies HBV inactive carriers Table 6. Accuracy of the ACE score cutoffs for identification of subjects who will be considered HBeAg-negative chronic infection (inactive carriers) during follow-up Total score <1 point ≤5 points ≤12 points ≤17 points Sensitivity, % (95% CI) 64.8 (59.0–70.1) 71.9 (66.4–76.8) 98.2 (95.9–99.2) 99.3 (97.5–99.8) Specificity, % (95% CI) 97.5 (87.1–99.6) 92.5 (80.4–97.4) 62.5 (47.0–75.8) 52.5 (37.5–67.1) Negative predictive value, % (95% CI) 28.3 (21.4–36.3) 31.9 (24.1–40.9) 83.3 (66.4–92.7) 91.3 (73.2–97.6) Positive predictive value, % (95% CI) 99.5 (97.0–99.9) 98.5 (95.8–99.5) 94.9 (91.7–96.9) 93.6 (90.3–95.9) Diagnostic accuracy, % (95% CI) 68.9 (63.6–73.7) 74.5 (66.4–78.9) 93.8 (90.6–95.9) 93.5 (90.2–95.7) CI, confidence interval. in the package of care of international health units, as well Ethical statement as of the need of predictive models that include a diverse genotype composition. Our study has some limitations because of the partially Ethical approval was obtained from the Ethical Commit- retrospective design, which could lead to bias resulting from tee of the University Hospital Vall d’Hebron (Code PR(AG) missing parameters such as HBcrAg and qHBsAg in some 247/2018; Date of approval 20 July 2018). patients. Furthermore, as previously mentioned, the pro- portion of patients with CHB was relatively low, but was Data sharing statement in line with the prevalence shown by other studies carried out in Europe.11,33 The external validation of our findings is limited by the unicentric character of the study, although The data used to support the findings of this study are avail- patients were followed up by different hepatologists. Also, able from the corresponding author upon request. a limited number of Asian and Hispanic subjects were in- cluded in the cohort. 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