Accepted Manuscript

Serum Hepatitis B core-related antigen is more accurate than HBsAg to identify

inactive carriers, regardless of HBV genotype

Mar Riveiro-Barciela, Marta Bes, Francisco Rodríguez-Frías, David Tabernero, Alicia

Ruiz, Rosario Casillas, Judith Vidal, Maria Homs, Leonardo Nieto, Silvia Sauleda,
Rafael Esteban, Maria Buti
PII: S1198-743X(17)30152-0
DOI: 10.1016/j.cmi.2017.03.003
Reference: CMI 887

To appear in: Clinical Microbiology and Infection

Received Date: 12 September 2016

Revised Date: 24 February 2017
Accepted Date: 5 March 2017

Please cite this article as: Riveiro-Barciela M, Bes M, Rodríguez-Frías F, Tabernero D, Ruiz A,
Casillas R, Vidal J, Homs M, Nieto L, Sauleda S, Esteban R, Buti M, Serum Hepatitis B core-related
antigen is more accurate than HBsAg to identify inactive carriers, regardless of HBV genotype, Clinical
Microbiology and Infection (2017), doi: 10.1016/j.cmi.2017.03.003.

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Intended category: virology.

Title: Serum Hepatitis B core-related antigen is more accurate than HBsAg to

identify inactive carriers, regardless of HBV genotype

Authors: Mar Riveiro-Barciela1,2, Marta Bes2,3, Francisco Rodríguez-Frías2,4, David

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Tabernero2,4, Alicia Ruiz2,4, Rosario Casillas5, Judith Vidal1, Maria Homs2,4, Leonardo

Nieto4, Silvia Sauleda2,3, Rafael Esteban1,2, Maria Buti1,2

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1. Liver Unit, Department of Internal Medicine, Hospital Universitari Vall d’Hebron

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and Universitat Autònoma de Barcelona, Barcelona, Spain

2. Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas

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(CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
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3. Transfusion Safety Laboratory, Banc de Sang i Teixits, Servei Català de la Salut,
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Barcelona, Spain
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4. Liver Pathology Unit, Departments of Biochemistry and Microbiology (Virology

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Unit) Hospital Universitari Vall d’Hebron and Universitat Autònoma de Barcelona,

Barcelona, Spain
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5. Vall d’Hebron Institut de Recerca, Barcelona, Spain

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Running title: HBcrAg to identify inactive carriers, regardless of genotype

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Corresponding author: Dr. Mar Riveiro-Barciela. Hospital Universitari Vall d’Hebron

Passeig Vall Hebron, 119-129. 08035, Barcelona, Spain. Telephone: (+34) 932746559,

Fax: (+34) 934274495, e-mail: mar.riveiro@gmail.com

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ABSTRACT

Objectives: to investigate whether hepatitis B surface antigen (HBsAg) and hepatitis B

core-related antigen (HBcrAg) levels are useful to identify inactive carriers among

HBeAg-negative patients infected by different HBV genotypes.

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Methods: 202 consecutive HBeAg-negative chronic hepatitis B patients, 135 inactive

carriers and 67 with HBV activity, were prospectively followed during 1 year.

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Results: In HBeAg-negative patients, HBsAg levels differed across the different

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genotypes (p<0.001). The highest levels were observed in genotypes F or H (4.2±0.6

logIU/mL), followed by genotype E (3.4±1.1 logIU/mL), A (3.4±0.8 logIU/mL), and

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the lowest in genotype D (2.7±1.1 logIU/mL). Variations in HBsAg levels were similar
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in inactive carriers and patients with HBV activity. HBsAg <3 logIU/mL showed good
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performance for identifying genotype-D inactive carriers: 76% of genotype D inactive

carriers met this cut-off versus ≤31% for genotypes A, E, F or H. However, in patients
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with genotype A, HBsAg levels ≤3.7 logIU/mL better classified inactive carriers. The
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combination of a single measurement of HBcrAg ≤3 logU/mL plus HBV DNA ≤2000

IU/mL yielded a positive predictive value and diagnostic accuracy >85% in all HBV
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genotypes, except genotype H or F, with values of 62.5% and 72.7%, respectively, for
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the two parameters.

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Conclusions: HBsAg levels varied across genotypes in HBeAg-negative patients.

HBsAg levels <3 log IU/mL were only useful for identifying genotype D inactive

carriers. A single HBcrAg measurement ≤3 logU/mL plus HBV DNA ≤2000 IU/mL

was highly accurate for identifying inactive carriers, regardless of their HBV genotype.
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INTRODUCTION

Hepatitis B e antigen (HBeAg)-negative chronic hepatitis B (CHB) infection is the most

common form of hepatitis B virus (HBV) infection in many countries, including

southern Europe [1, 2]. One of the most challenging issues in CHB is to distinguish

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between the active HBeAg-negative phase of the infection and the inactive carrier state.

The prognosis of the disease and indication for therapy differ in these two clinical

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situations; hence, their differentiation is crucial. Therapy is not recommended in

HBeAg-negative inactive carriers, and monitoring can be reduced because of their

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favourable long-term prognosis. In contrast, HBeAg-negative patients with HBV

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activity have a higher risk of developing liver fibrosis and hepatocellular carcinoma, so
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regular monitoring and in many cases, antiviral therapy are needed. To accurately

identify inactive carrier status, the current guidelines recommend at least 3 alanine
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aminotransferase (ALT) and HBV DNA determinations during 1 year.

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Since Blumberg’s discovery of hepatitis B surface antigen (HBsAg) in 1965, this

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parameter has been accepted as a laboratory marker to establish HBV infection [3].

HBsAg production is controlled, at least in part, by the amount of intrahepatic

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covalently closed circular DNA (cccDNA) and the specific host immune response

against the HBV envelope proteins. This is particularly evident in HBeAg-positive

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patients who show a high correlation between cccDNA and HBsAg levels [4]. The
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amount of circulating HBsAg varies during the different phases of chronic infection:

levels are higher in immunotolerant patients and they decrease after HBeAg

seroconversion, and levels are lower in inactive carriers than in HBeAg-negative CHB

patients [5-8]. For this reason, HBsAg determination has been cited as a potentially

useful tool to identify inactive carriers [9]. Brunetto et al [10] proposed a combination

of HBsAg level <3 logIU/mL plus HBV DNA ≤2000 IU/mL to identify inactive carrier
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status at a single time point in HBeAg-negative patients. The reported diagnostic

accuracy was 93%, but the cohort was limited to genotype D-infected patients.

Currently, 10 different HBV genotypes have been described, each with a characteristic

geographical distribution [11, 12]: genotype A predominates in Northern Europe and

America, genotype B and C in Asia, genotype D in southern Europe, genotype E in

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Africa, and genotype H and F in South America [2, 12]. Recently, hepatitis B core-

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related antigen (HBcrAg) has emerged as a new serological marker for CHB. As

HBcrAg correlates well with intrahepatic cccDNA levels in both naïve patients and

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those treated with nucleoside analogues (NUCs) [13, 14], it may also be useful for

proper classification of HBeAg-negative patients.

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The aim of this study was to investigate the performance of HBsAg and HBcrAg

determinations to categorize HBeAg-negative patients across the various HBV

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genotypes.
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PATIENTS AND METHODS

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Patient Selection and definitions

Consecutive treatment-naïve HBeAg-negative patients chronically infected with HBV

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(HBsAg-positive for more than 6 months) were prospectively selected in the outpatient
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clinics of a tertiary hospital. According to the guidelines of the European Association

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for the Study of the Liver (EASL) [9], patients were classified as follows: inactive

carriers, persistently normal ALT levels and HBV DNA ≤2000 IU/mL in 3

determinations over 1 year; active carriers (AC), HBV DNA >20,000 IU/mL, or at least

1-time fluctuation of HBV DNA >2000 IU/mL plus abnormal ALT levels (above the

upper limit of normality, ULN). Patients were regarded as “intermediate” on HBV DNA

fluctuation between 2000 and 20,000 IU/mL regardless of ALT levels within the year of
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follow-up [15]. For the purposes of this study, patients classified as either intermediate

or AC were analysed together as patients with HBV activity, since all had HBV DNA

>2000 IU/mL and therefore, a 3-fold higher probability of disease progression [9, 16].

Patients were excluded if they had undergone liver transplantation, were co-infected

with hepatitis C virus, hepatitis D virus, or human immunodeficiency virus (HIV), had

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high alcohol intake, evidence of liver cirrhosis based on ultrasound findings (hepatic

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parenchyma nodules, spleen >12 cm, portal vein >16 mm), and/or analytical data

(platelet count persistently below 140x10E9/mL). This study was conducted in

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accordance with the Declaration of Helsinski guidelines and the principles of Good

Practise, and was approved by the Ethic Review Board of the Vall d’Hebron Hospital.

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Data Acquisition

Data on demographics (sex, age, and race) were prospectively collected, and the clinical
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history was collected from the patients’ medical records at the time of enrolment. Data
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on chemistry (ALT, aspartate aminotransferase [AST], gamma-glutamyl transferase,

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bilirubin, and albumin), haematology (platelet count, prothrombin time), and HBV

serology and virology (serum HBsAg and HBcrAg, HBV DNA, HBV genotype) were
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also collected.

Laboratory Measurements
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HBsAg was quantified using the COBAS 80000 HBsAg II assay (Roche Diagnostics,

Mannheim, Germany): lower limit of detection, 0.05 IU/mL. HBcrAg was quantified by

an electrochemiluminescent assay: Lumipulse® G HBcrAg assay (Fujirebio, Fujirebio

Europe, Gent, Belgium): lower limit of detection, 2 logU/mL. This technique

simultaneously determines denatured HBeAg, HBcAg, and a 22 kDa precore protein

(p22cr), as all three share an identical sequence of 149 amino acids. Hepatitis C,
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hepatitis D, and HIV were detected by commercially available immunoassays. Serum

HBV DNA was quantified by PCR with a COBAS 6800 HBV test (Roche Diagnostics,

Mannheim, Germany): lower limit of quantification, 20 IU/mL and lower limit of

detection, 10 IU/mL and lower limit of detection, 3.6 IU/mL. For HBV genotyping,

HBV DNA was first enriched by ultracentrifugation of 9.6 mL of serum. Subsequently,

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Sanger sequencing was carried out after amplification of two different viral regions:

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PreC/Core (nucleotides 1774-2389, 615 bp) and PreS/Surface (nucleotides 2828-176,

561 bp), as previously reported [17]. Phylogenetic analysis was performed with HBV

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reference sequences using Neighbor-Joining analysis with the MEGA program, version

6. Genotypes H and F were combined due to their phylogenetic proximity and

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geographic distribution in the same areas [18, 19].
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Statistical Analysis
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Normally distributed quantitative variables were compared with the Student t test and

expressed as the mean ± standard deviation (SD). Variables with a non-normal

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distribution were analysed with the Mann-Whitney U test and expressed as the median
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and interquartile range. Categorical variables were compared between groups using the
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Chi-square or Fisher exact test, as appropriate. Correlations were tested with the

Spearman correlation test. The diagnostic performance of HBsAg and HBcrAg levels
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was evaluated by receiver operating characteristic (ROC) curve analysis. The cut-off
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value to differentiate inactive carriers from patients with HBV activity was selected

considering the highest Youden index. All statistical analyses were performed using

IBM SPSS, 20 (SPSS Inc., Chicago, USA).

RESULTS

Baseline characteristics
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Of the 213 consecutive treatment-naïve HBeAg-negative patients evaluated, 202 were

included: 135 (66.8%) classified as inactive carriers, 12 (6%) as ACs, and 55 (27%) as

intermediates (Supplementary Figure 1). Therefore, 67 individuals met the HBV activity

criteria. Eleven patients (5.2%) with HBV DNA <10 IU/mL were excluded because

HBV genotype could not be determined at this level.

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Baseline characteristics are summarized in Table 1 and Supplementary Table S1. The

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predominant genotypes were A (37%), D (29%), E (18%), and F or H (12%). Patients

having HBV activity were younger, and had higher ALT, HBV DNA, HBsAg, and

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HBcrAg levels than inactive carriers (Table 1). Distribution of HBV genotypes was

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similar between inactive carriers and patients with HBV activity (p=0.98). Overall,
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mean HBV DNA levels were similar between the main genotypes (p=0.91). Univariate

and multivariate analyses of baseline characteristics between inactive carriers and

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patients with HBV activity are summarized in Supplementary Table S2.

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Serum HBsAg levels according to the phase of chronic hepatitis B infection and
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HBV genotype

Overall, HBsAg levels were lower in inactive carriers than in patients with HBV
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activity (p=0.003). HBsAg levels statistically differed (p<0.001) across the HBV
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genotypes, being highest for genotype F or H, followed by E, A, and the lowest, D

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(Table 2). When HBsAg levels were compared between these two groups according to

HBV GT, significant differences were only observed for genotypes A and D (p=0.002

and p<0.001, respectively) (Figure 1).

A positive correlation between HBsAg and HBV DNA was found in patients with HBV

genotype A (r= 0.46, p<0.001) and D (r= 0.56, p<0.001) infection, but there were no

correlations in genotypes E, or F or H (Figure 2a).

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Serum HBcrAg levels according to the phase of chronic hepatitis B infection and

HBV genotype

HBcrAg levels varied among the HBV genotypes in patients with HBV activity

(p=0.02) (Table 2) and were similar in inactive carriers (p=0.052) (Supplementary Table

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S3). In the comparison of HBcrAg levels between inactive carriers and patients with

HBV activity according to genotype, differences were only observed in genotype A-

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infected patients (Figure 1). Correlations between HBcrAg and HBV DNA were only

observed in genotype A (r= 0.43, p<0.001), although there was a trend to a correlation

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in genotype E patients (r=0.49, p=0.057) (Figure 2b). All HBV genotype H and F

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patients had HBcrAg levels below 2 logU/mL, the lower limit of detection, which
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precluded the study of correlations.

There was a moderate correlation between HBsAg and HBcrAg levels in patients with
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genotypes A (r=0.55, p<0.001) and D (r=0.41, p=0.01), and no correlations in genotype

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E, F, or H infection (Figure 2b).

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HBsAg and HBcrAg levels to identify HBV inactive carriers

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Overall, the area under the receiver operating characteristic curve (AUC) for

identification of inactive carrier status was 0.67 (95% CI, 0.56-0.77; p=0.003) for
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HBcrAg and 0.63 (95% CI, 0.55-0.71; p=0.001) for HBsAg. The cut-off associated with
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the highest Youden index was 3.21 logIU/mL for HBsAg (sensitivity, 49.6%;

specificity, 77.6%) and 3 logU/mL for HBcrAg (sensitivity, 97.84%; specificity,

27.3%). In genotype A, the HBcrAg AUC was higher than that of HBsAg (AUC 0.80,

95% CI 0.65-0.95; p<0.001, and AUC 0.73, 95% CI 0.60-0.85; p=0.002, respectively).

In genotype D, only HBsAg determination had sufficient discriminatory power to

identify inactive carriers (AUC 0.78, 95% CI 0.66-0.90; p<0.001). Higher HBsAg levels
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in genotype A than genotype D-infected patients at baseline resulted in different cut-offs

to identify inactive carriers. HBsAg <3 logIU/mL showed a diagnostic accuracy of

74.1% for genotype D, but only 49.3% for genotype A (Table 3). The cut-off showing

the best performance in genotype A was HBsAg ≤3.7 logIU/mL (positive predictive

value [PPV] 82.2%; negative predictive value [NPV] 50%; diagnostic accuracy 69.3%).

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However, the previous cut-offs were not useful for proper classification of inactive

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carriers infected by other HBV genotypes (genotype E, AUC 0.57; p=0.48 and genotype

F or H, AUC 0.49; p=0.95).

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The diagnostic accuracy of HBcrAg determination was higher than that of HBsAg for

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identifying inactive carriers. A single HBV DNA determination ≤2000 IU/mL together
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with HBcrAg ≤3 logU/mL yielded a PPV and diagnostic accuracy >85% for all

genotypes except H or F, which showed values of 63% and 73%, for these two indexes,
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respectively (Table 3). Inclusion of 11 patients with undetectable HBV DNA in the

cohort did not change the diagnostic accuracy or predictive values of the above-
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mentioned cut-offs (Supplementary Table S4). HBsAg determination with or without

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HBV DNA values did not achieve higher diagnostic accuracy than the combined cut-
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offs of HBcrAg ≤3 logU/mL plus HBV DNA ≤2000 IU/mL.

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Variability of HBsAg levels

In total, 103 (51%) and 81 (40%) patients had consecutive quantitative HBsAg

determinations separated by 6 and 12 months, respectively. The comparisons showed no

differences in HBsAg levels over this time period (month 6: mean difference 0.036,

95%CI -0.035 to 0.11; p=0.32; month 12: mean difference 0.069, 95%CI -0.024 to 0.16;

p=0.15). In the comparison of consecutive HBsAg levels in inactive carriers, 48 patients

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had at least 2 determinations separated by 12 months, and a significant decay in HBsAg

levels was observed in this group (mean difference 0.16, 95%CI 0.078-0.23; p<0.001).

However, in the 33 patients with HBV activity and an HBsAg follow-up of at least 1

year, no changes were observed (p=0.67).

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DISCUSSION

In this prospective study of well-classified HBeAg-negative patients infected by HBV

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genotypes A and D to F, we found considerable variability in HBsAg levels across the

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genotypes, with statistically higher levels in patients with genotype F or H, followed by

E, A and D. There was a high correlation between HBsAg and HBV DNA levels in

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genotype A and D patients, which made HBsAg quantification a good biomarker for
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proper identification of inactive carriers infected by either of these genotypes. Our

results confirmed that the previously described HBsAg cut-off of <3 logIU/mL [10] is
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particularly useful to identify genotype D inactive carriers, with a positive predictive

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value of 76% and diagnostic accuracy of 74%. These results stem from the low HBsAg
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levels in patients with genotype D, the genotype associated with the lowest levels (mean

2.7 logIU/mL). In the remaining HBV genotypes, the percentage of inactive carriers
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who met this criterion was ≤31%, a finding that indicates the need for HBV genotyping

to use this HBsAg cut-off value. For example, in genotype A patients, an HBsAg value
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of ≤3.7 logIU/mL yielded the best performance for identifying inactive carriers: positive
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predictive value, 82% and diagnostic accuracy, 69%.

HBcrAg is a recently described serum marker formed by 3 products from the

preCore/Core gene that have an identical sequence of 149 amino acids. As HBcrAg

level decreases across the different phases of chronic hepatitis B, it has been suggested

as a potential tool for identifying patients at risk of developing hepatocellular carcinoma

or HBV reactivation after discontinuing NUCs [20, 21]. There is scarce data on the
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possible role of HBcrAg levels to help classify the chronic hepatitis B infection stage. In

our cohort, HBcrAg levels were associated with excellent predictive values for

identifying HBV inactive carriers, in particular when a single measurement of this

biomarker was combined with HBV DNA ≤2000 IU/mL. HBcrAg levels achieved

higher diagnostic accuracy for classifying inactive carriers across all the HBV

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genotypes than HBsAg quantification: a single HBV DNA determination ≤2000 IU/mL

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plus HBcrAg levels of ≤3 logU/mL showed an overall PPV and diagnostic accuracy of

>85%.

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There is extensive information concerning HBsAg levels in HBV genotypes B and C [5,

7, 22-24]. However, data are scarce for genotype E to H, the most common genotypes in

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developing countries, where HBV is more prevalent [18, 19, 25, 26]. Since HBV DNA
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monitoring is not currently available worldwide [27], it would be useful to have a
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serological marker that can identify inactive carriers in a single determination with less

sophisticated technology and easier manipulation and preservation of serum samples [9].
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HBsAg quantification could be a good candidate, but the significant variation in HBsAg
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levels across the HBV genotypes indicates that genotyping would be needed for proper

classification of inactive carriers. High HBsAg levels were also reported in a cohort of
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128 HBeAg-negative HIV-HBV co-infected patients with HBV genotype E (mean 3.82

logIU/mL) [28] and in a European cohort of HBV patients, where higher HBsAg levels
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were reported in genotype A than in genotype D (p=0.007) [6]. However, this difference

was not seen in a small number of inactive carriers (8 A and 18 D). Similar results have

been described in cell cultures. Sugiyama et al showed that extracellular expression of

viral DNA and surface antigens differs between genotypes, with higher HBsAg levels in

genotype A than genotype D [29]. Moreover, HBsAg levels have a tendency to decay

over a 1-year period in inactive carriers, whereas they remain stable in patients with
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HBV activity. In a retrospective multicenter study including 292 HBeAg-negative

patients, the mean annual HBsAg decrease was more prominent in inactive carriers than

in those with HBV activity [15].

A correlation between HBcrAg levels and HBV DNA has been reported in HBeAg-

negative genotype A and D patients [30], but there are no available data for genotypes

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E, F or H. In the present study, the correlations between HBV DNA and both HBsAg

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and HBcrAg are lower than previously reported values [30, 31]. This may be because

only HBeAg-negative patients were enrolled on our study, and most were inactive

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carriers with HBV DNA persistently ≤2000 IU/mL.

Our study has several limitations, one of them being the heterogeneous distribution of

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HBV genotypes. This occurred because patients were unselected and prospectively
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included, and that implied a higher percentage of the more prevalent HBV genotypes in
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our area (A and D). Another limitation is that HBcrAg could not be determined in a

small number of patients. Therefore, the use of HBcrAg for proper classification of
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inactive carriers regardless of HBV genotype should be validated in a larger cohort. One
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of the strengths of our study is inclusion of patients with less extensively investigated

HBV genotypes, such as E, F and H, in whom data in this regard are scarce.
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The results of this study could have implications for future research. The variability of

HBsAg levels among HBeAg-negative patients with different genotypes should be

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taken into account when this serum marker is used for clinical purposes.

In conclusion, in a large cohort of well-categorized HBeAg-negative patients with

chronic HBV infection, HBsAg levels varied among inactive carriers according to

genotype, with higher levels in genotype F or H. In contrast, HBcrAg levels were

similar regardless of the genotype, and when combined with HBV DNA determination,

HBcrAg enabled identification of inactive carrier status without HBV genotyping.

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FUNDING

This study was funded in part by the Instituto de Salud Carlos III (grant number

PI12/01893) and from Gilead (grant number GLD13/00137)

CONFLICTS OF INTEREST

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María Buti and Rafael Esteban have served as advisors to Gilead, Bristol-Myers Squibb

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and Novartis. Maria Buti and Mar Riveiro-Barciela have received research grants from

Gilead. The other authors have no personal interests to declare

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ACKNOWLEDGEMENTS
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HBcrAg reagents were provided free of charge by Fujirebio Europe. The funder had no

role in the study design, data collection, analysis or preparation of the manuscript.
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English writing support was provided by Celine Cavallo.

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AUTHORSHIP
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Maria Buti (MB) and Francisco Rodriguez-Frias act as guarantors of this article. MB,

Rafael Esteban (RE) and Mar Riveiro-Barciela (MRB) designed the study. MRB, Marta
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Bes (MB) and Silvia Sauleda (SS) selected the patients. Maria Homs (MH) and David
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Tabernero (DT) performed quantification of HBsAg and HBV DNA. Rosario Casillas

(RC), María Blasi (MB) and Leonardo Nieto (LN) carried out HBV DNA amplification

and genotyping. MRB, MB and Judith Vidal (JV) collected the clinical and laboratory

data. MH, DT, LN and FRF analysed and interpreted the data. MRB, MB and FRF

drafted the manuscript. MB, RE and FRF reviewed the manuscript. All authors

approved the final version of the article.

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therapy. Hepatology research : the official journal of the Japan Society of Hepatology.
2007; 37: 661-666.
21 Tada T, Kumada T, Toyoda H, et al. Hbcrag predicts hepatocellular carcinoma
development: An analysis using time-dependent receiver operating characteristics.
Journal of hepatology. 2016; 65: 48-56.
22 Seto WK, Wong DK, Fung J, et al. A large case-control study on the predictability of
hepatitis b surface antigen levels three years before hepatitis b surface antigen

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seroclearance. Hepatology (Baltimore, Md). 2012; 56: 812-819.
23 Tseng TC, Liu CJ, Chen CL, et al. Higher lifetime chance of spontaneous surface antigen
loss in hepatitis b carriers with genotype c infection. Alimentary pharmacology &
therapeutics. 2015; 41: 949-960.

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24 Tseng TC, Liu CJ, Su TH, et al. Serum hepatitis b surface antigen levels predict surface
antigen loss in hepatitis b e antigen seroconverters. Gastroenterology. 2011; 141: 517-
525, 525.e511-512.

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25 Kidd-Ljunggren K, Miyakawa Y, Kidd AH. Genetic variability in hepatitis b viruses. The
Journal of general virology. 2002; 83: 1267-1280.
26 Okamoto H, Tsuda F, Sakugawa H, et al. Typing hepatitis b virus by homology in

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nucleotide sequence: Comparison of surface antigen subtypes. The Journal of general
virology. 1988; 69 ( Pt 10): 2575-2583.
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27 Who guidelines approved by the guidelines review committee. Guidelines for the
prevention, care and treatment of persons with chronic hepatitis b infection. Geneva:
World Health Organization Copyright (c) World Health Organization 2015. 2015.
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28 Boyd A, Maylin S, Moh R, et al. Hepatitis b surface antigen quantification as a predictor

of seroclearance during treatment in hiv-hepatitis b virus co-infected patients from
sub-saharan africa. Journal of gastroenterology and hepatology. 2015.
29 Sugiyama M, Tanaka Y, Kato T, et al. Influence of hepatitis b virus genotypes on the
D

intra- and extracellular expression of viral DNA and antigens. Hepatology (Baltimore,
Md). 2006; 44: 915-924.
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30 Maasoumy B, Wiegand SB, Jaroszewicz J, et al. Hepatitis b core-related antigen

(hbcrag) levels in the natural history of hepatitis b virus infection in a large european
cohort predominantly infected with genotypes a and d. Clinical microbiology and
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infection : the official publication of the European Society of Clinical Microbiology and
Infectious Diseases. 2015; 21: 606.e601-610.
31 Seto WK, Wong DK, Fung J, et al. Linearized hepatitis b surface antigen and hepatitis b
core-related antigen in the natural history of chronic hepatitis b. Clinical microbiology
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and infection : the official publication of the European Society of Clinical Microbiology
and Infectious Diseases. 2014; 20: 1173-1180.
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FIGURE LEGENDS

Figure 1. Comparison of mean hepatitis B surface antigen (HBsAg) and hepatitis B

core-related antigen (HBcrAg) levels between inactive carriers and patients with HBV

activity showed statistical differences in patients infected by genotype A (HBsAg and

HBcrAg) and D (HBsAg), but not in those with GT E, F or H infection.

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Figure 2. Correlations between HBV DNA and both hepatitis B surface antigen

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(HBsAg) (a) and hepatitis B core-related antigen (HBcrAg) (b) reached statistical

significance. Independent analysis by each genotype showed that correlations were only

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present in patients infected by genotype A (HBsAg and HBcrAg) and D (only HBsAg).
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Moderate correlations between HBsAg and HBcrAg levels (c) were found in genotype
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A and D patients.
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Table 1. Baseline characteristics according to the stage of HBV infection.

Total Inactive carriers HBV activity

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N= 202 N=135 N= 67 p value

Age, years, median(IQR) 46 (36-56) 48 (39-57) 43 (32-54) 0.004

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Male, n (%) 112 (55%) 81 (60%) 31(47%) 0.07

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Genotype, n (%)

-A 75 (37%) 52 (38%) 23 (34%)

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- B or C 5 (2%) 1 (1%) 4(6%) 0.98

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-D 58 (29%) 38 (28%) 20 (30%)

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-E 36 (18%) 26 (19%) 10 (15%)

- F or H 24 (12%) 16 (12%) 8 (12%)

D
- Mixed A/E 4 (2%) 2 (2%) 2 (3%)

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ALT, IU/mL, median(IQR) 18 (13-28) 16 (11-24) 23 (16-33) <0.001
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Platelets, 10E9/L, median(IQR) 225 (188-251) 222 (185-248) 227 (197-255) 0.25

Albumin, mg/dL, median(IQR) 4.4 (4.2-4.6) 4 (4.2-4.6) 4.4 (4.1-4.6) 0.81

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HBsAg, logIU/mL, median(IQR) 3.5 (2.6-4.1) 3.3 (2.4-4.0) 3.7 (3.2-4.1) 0.003
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HBcrAg, logU/mL, median(IQR)€ 2.0 (2-2.5) 2.0 (2.0-2.0) 2.0 (2.0-3.3) <0.001

HBV DNA, logIU/mL, median(IQR) 2.7 (2.1-3.3) 2.5 (1.9-2.9) 3.5 (3.0-3.8) <0.001
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Available in 134 patients
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Table 2. Comparison of HBsAg and HBcrAg levels according to HBV genotype and disease activity in HBV infection

Total GT A GT D GT E GT F or H p value

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N 202 75 58 36 24 -

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Total
HBsAg, logIU/mL, median(IQR) 3.5(2.6-4.1) 3.5(3.1-4.0) 2.7(2.1-3.5) 3.7(3.0-4.2) 4.3(4.1-4.4) p<0.001

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HBcrAg, logU/mL, median(IQR) 2.0(2.0-2.5) 2.0(2.0-2.7) 2.0(2.0-2.0) 2.0(2.0-2.5) 2.0(2.0-2.0) 0.052

N(%) 135(67%) 52(39%) 38(28%) 26(19%) 16(12%) -

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Inactive

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carriers HBsAg, logIU/mL, median(IQR)& 3.3(2.4-4.0) 2.7(3.3-3.8) 2.4(1.8-2.9) 3.7(2.9-4.2) 4.3(3.9-4.5) <0.001

HBcrAg, logU/mL, median(IQR)± 2.0(2.0-2.0) 2.0(2.0-2.2) 2.0(2.0-2.0) 2.0-2.0-2.3) 2.0(2.0-2.0) 0.63

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N(%) 67(33%) 23(34%) 20(30%) 10(15%) 8(12%) -

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HBsAg, logIU/mL, median(IQR)£

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3.7(3.2-4.1) 3.8(3.5-4.2) 3.4(2.9-3.7) 3.8(3.2-4.2) 4.3(4.2-4.4) 0.003
HBV activity
HBcrAg, logU/mL, median(IQR)¥ 2.0(2.0-3.3) 2.9(2.0-3.4) 2.0(2.0-2.6) 2.0(2.3-5.8) 2.0(2.0-2.0) 0.02
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HBsAg in inactive carriers: p<0.001 for genotype A vs. D; p=0.57 for genotype A vs. E; p<0.001 for genotype A vs. F or H; p<0.001 for genotype D vs. E; p<0.001 for
genotype D vs. F or H; p=0.013 for genotype E vs. F or H.
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HBcrAg in inactive carriers: p=0.55 for genotype A vs. D; p=0.75 for genotype A vs. E; p=0.24 for genotype A vs. F or H; p=0.52 for genotype D vs. E; p=0.48 for genotype
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D vs. F or H; p=0.14 for genotype E vs. F or H.

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HBsAg in patients with HBV activity: p=0.02 for genotype A vs. D; p=0.87 for genotype A vs. E; p=0.06 for genotype A vs. F or H; p=0.09 for genotype D vs. E; p<0.001
for genotype D vs. F or H; p=0.07 for genotype E vs. F or H.
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HBcrAg in HBV activity: p=0.03 for genotype A vs. D; p=0.94 for genotype A vs. E; p=0.01 for genotype A vs. F or H; p=0.34 for genotype D vs. E; p=0.21 for genotype D
vs. F or H; p=0.13 for genotype E vs. F or H.

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Table 3. Diagnostic accuracy and predictive values of HBsAg and HBcrAg cut-offs for identifying inactive carriers status according to HBV

genotype

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HBsAg <3 logIU/mL HBsAg ≤3.21 logIU/mL HBsAg ≤3.21 logIU/mL + HBV DNA ≤2000 IU/mL
Population Total GT A GT D GT E GT F or H Total GT A GT D GT E GT F or H Total GT A GT D GT E GT F or H
N=202 N=75 N=58 N=36 N=24 N=202 N=75 N=58 N=36 N=24 N=202 N=75 N=58 N=36 N=24

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PPV 55/69 16/18 29/35 8/10 1/1 67/82 24/26 31/38 10/12 1/1 67/75 24/26 31/33 10/11 1/1

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(80%) (89%) (83%) (80%) (100%) (82%) (92%) (82%) (83%) (100%) (89%) (92%) (94%) (91%) (100%)
NPV 53/133 21/57 14/23 8/26 8/23 52/120 21/49 13/20 8/24 8/23 59/127 21/49 18/25 9/25 8/23
(40%) (37%) (61%) (31%) (35%) (43%) (43%) (65%) (33%) (35%) (46%) (43%) (67%) (36%) (35%)

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Diagnostic 108/202 37/75 43/58 16/36 9/24 119/202 45/75 44/58 18/36 9/24 126/202 45/75 49/58 19/36 9/24
accuracy (53%) (49%) (74%) (44%) (38%) (59%) (60%) (76%) (50%) (38%) (62%) (60%) (84%) (53%) (38%)

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HBcrAg ≤3 logU/mL HBcrAg ≤3 logU/mL+ HBV DNA ≤2000 IU/mL

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Population Total GT A GT D GT E GT F or H Total GT A GT D GT E GT F or H
N=134 N=65 N=38 N=16 N=11 N=134 N=65 N=38 N=16 N=11

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PPV 88/120 47/57 24/35 11/14 5/11 88/103 47/52 24/28 11/12 5/8

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(73%) (82%) (69%) (79%) (46%) (86%) (90%) (86%) (92%) (63%)
NPV 12/14 7/8 2/3 2/2 0/0 29/31 12/13 9/10 4/4 3/3
(86%) (88%) (67%) (100%) (0%) (91%) (92%) (90%) (100%) (100%)
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Diagnostic 100/134 54/65 26/38 13/16 5/11 117/134 59/65 33/38 15/16 8/11
accuracy (75%) (83%) (68%) (81%) (46%) (87%) (91%) (87%) (94%) (73%)
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p=0.67 p=0.5

p=0.002 p=0.5

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p<0.001

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p<0.001

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p=0.21

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p=0.67

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Inactive carriers Inactive carriers

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HBV activity EP HBV activity

HBV genotype HBV genotype

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HBV DNA (logIU/mL)

r=0.30 r=0.46 r=0.56

Overall p<0.001 Genotype A p<0.001 Genotype D p<0.001

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r=0.17
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Genotype E p=0.33 Genotype H or F p=0.39
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HBsAg (logIU/mL)
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r=0.37 r=0.43 r=0.29
p<0.001 p<0.001 p=0.08

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HBV DNA (logIU/mL)

Overall Genotype A Genotype D

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r=0.49

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p=0.057

Genotype E Genotype H or F
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HBcrAg (logU/mL)
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HBsAg (logIU/mL)

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r=0.39 r=0.55 r=0.41
p<0.001 p<0.001 P=0.01

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Overall Genotype A Genotype D

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HBcrAg (logU/mL)

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