PIIS1198743X17301520

Clinical Microbiology and Infection 23 (2017) 860e867
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Clinical Microbiology and Infection
journal homepage: www.clinicalmicrobiologyandinfection.com
Original article
Serum hepatitis B core-related antigen is more accurate than hepatitis
B surface antigen to identify inactive carriers, regardless of hepatitis B
virus genotype
M. Riveiro-Barciela 1, 2, *, M. Bes 2, 3, F. Rodríguez-Frías 2, 4, D. Tabernero 2, 4, A. Ruiz 2, 4,
lez 1, M. Homs 2, 4, L. Nieto 4, S. Sauleda 2, 3, R. Esteban 1, 2,
R. Casillas 5, J. Vidal-Gonza
1, 2
M. Buti
noma de Barcelona, Barcelona, Spain
Liver Unit, Department of Internal Medicine, Hospital Universitari Vall d'Hebron and Universitat Auto
ticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain
n Biom
Centro de Investigacio
edica en Red de Enfermedades Hepa
3)
de la Salut, Barcelona, Spain
Transfusion Safety Laboratory, Banc de Sang i Teixits, Servei Catala
4)
noma de
Liver Pathology Unit, Departments of Biochemistry and Microbiology (Virology Unit) Hospital Universitari Vall d'Hebron and Universitat Auto
Barcelona, Barcelona, Spain
5)
Vall d'Hebron Institut de Recerca, Barcelona, Spain
1)
2)
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 September 2016
Received in revised form
24 February 2017
Accepted 5 March 2017
Available online 11 March 2017
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 hepatitis B virus (HBV) genotypes.
Methods: In all, 202 consecutive HBeAg-negative patients with chronic hepatitis B, 135 inactive carriers
and 67 with HBV activity, were prospectively followed for 1 year.
Results: In HBeAg-negative patients, HBsAg levels differed across the different 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), genotype A (3.4 ± 0.8 logIU/mL), and the lowest in genotype D (2.7 ± 1.1 logIU/mL).
Variations in HBsAg levels were similar in inactive carriers and patients with HBV activity. HBsAg
<3 logIU/mL showed good 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 with genotype A, HBsAg levels 3.7 logIU/mL better classiﬁed inactive carriers. The 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 genotypes, except genotype H or F, with values of 62.5% and 72.7%,
respectively, for the two parameters.
Conclusions: HBsAg levels varied across genotypes in HBeAg-negative patients. HBsAg levels <3 logIU/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. M. Riveiro-Barciela, Clin Microbiol Infect 2017;23:860
© 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All
rights reserved.
Editor: G. Antonelli
Keywords:
HBcrAg
HBeAg negative chronic hepatitis B
HBsAg
HBV inactive carrier
Hepatitis B
Hepatitis B virus genotype
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
* Corresponding author. M. Riveiro-Barciela, Hospital Universitari Vall d'Hebron,
Passeig Vall Hebron, 119-129, 08035, Barcelona, Spain.
E-mail address: [email protected] (M. Riveiro-arciela).
the most challenging issues in CHB is to distinguish between the
active HBeAg-negative phase of the infection and the inactive carrier state. The prognosis of the disease and the indication for
therapy differ in these two clinical situations; hence, their differentiation is crucial. Therapy is not recommended in HBeAgnegative inactive carriers, and monitoring can be reduced
because of their favourable long-term prognosis. In contrast,
HBeAg-negative patients with HBV activity have a higher risk of
http://dx.doi.org/10.1016/j.cmi.2017.03.003
1198-743X/© 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
M. Riveiro-Barciela et al. / Clinical Microbiology and Infection 23 (2017) 860e867
developing liver ﬁbrosis and hepatocellular carcinoma, so regular
monitoring and, in many cases, antiviral therapy are needed. To
accurately identify inactive carrier status, the current guidelines
recommend at least three alanine aminotransferase (ALT) and HBV
DNA determinations during 1 year.
Since Blumberg's discovery of hepatitis B surface antigen
(HBsAg) in 1965, this 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 covalently
closed circular DNA and the speciﬁc host immune response against
the HBV envelope proteins. This is particularly evident in HBeAgpositive patients who show a high correlation between covalently
closed circular DNA and HBsAg levels [4]. The 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 [5e8]. 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 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, ten
different HBV genotypes have been described, each with a characteristic geographical distribution [11,12]: genotype A predominates in northern Europe and America, genotypes B and C in
Asia, genotype D in southern Europe, genotype E in Africa, and
genotypes H and F in South America [2,12]. Recently, hepatitis B
core-related antigen (HBcrAg) has emerged as a new serological
marker for CHB. As HBcrAg correlates well with intrahepatic
covalently closed circular DNA levels in both naïve patients and
those treated with nucleoside analogues [13,14], it may also be
useful for proper classiﬁcation of HBeAg-negative patients.
The aim of this study was to investigate the performance of
HBsAg and HBcrAg determinations to categorize HBeAg-negative
patients across the various HBV genotypes.
Patients and methods
Patient selection and deﬁnitions
Consecutive treatment-naive HBeAg-negative patients chronically infected with HBV (HBsAg-positive for more than 6 months)
were prospectively selected in the outpatient clinics of a tertiary
hospital. According to the guidelines of the European Association
for the Study of the Liver [9], patients were classiﬁed as follows:
inactive carriers, persistently normal ALT levels and HBV DNA
2000 IU/mL in three determinations over 1 year; active carriers,
HBV DNA >20 000 IU/mL, or at least one ﬂuctuation of HBV DNA
>2000 IU/mL plus abnormal ALT levels (above the upper limit of
normality). Patients were regarded as ‘intermediate’ on HBV DNA
ﬂuctuation between 2000 and 20 000 IU/mL regardless of ALT
levels within the year of follow up [15]. For the purposes of this
study, patients classiﬁed as either intermediate or active carriers
were analysed together as patients with HBV activity, as all had HBV
DNA >2000 IU/mL and therefore, a three-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 high alcohol
intake, evidence of liver cirrhosis based on ultrasound ﬁndings
(hepatic parenchyma nodules, spleen >12 cm, portal vein >16 mm),
or analytical data (platelet count persistently below 140 10E9/
mL). This study was conducted in accordance with the Declaration
of Helsinki guidelines and the principles of Good Practice, and
861
was approved by the Ethic Review Board of the Vall d'Hebron
Hospital.
Data acquisition
Data on demographics (sex, age and race) were prospectively
collected, and the clinical history was collected from the patients'
medical records at the time of enrolment. Data on chemistry (ALT,
aspartate aminotransferase (AST), g-glutamyl transferase, bilirubin,
and albumin), haematology (platelet count, prothrombin time), and
HBV serology and virology (serum HBsAg and HBcrAg, HBV DNA,
HBV genotype) were also collected.
Laboratory measurements
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 virus, hepatitis D
virus and HIV were detected by commercially available immunoassays. Serum HBV DNA was quantiﬁed by PCR with a COBAS 6800
HBV test (Roche Diagnostics): lower limit of quantiﬁcation 20 IU/
mL, lower limit of detection 10 IU/mL. For HBV genotyping, HBV
DNA was ﬁrst enriched by ultracentrifugation of 9.6 mL of serum.
Subsequently, Sanger sequencing was carried out after ampliﬁcation of two different viral regions: PreC/Core (nucleotides
1774e2389, 615 bp) and PreS/Surface (nucleotides 2828e176,
561 bp), as previously reported [17]. Phylogenetic analysis was
performed with HBV reference sequences using neighbour-joining
analysis with the MEGA program, version 6. Genotypes H and F
were combined due to their phylogenetic proximity and
geographic distribution in the same areas [18,19].
Statistical analysis
Normally distributed quantitative variables were compared
with the Student's t test and expressed as the mean ± standard
deviation. Variables with a non-normal distribution were analysed
with the ManneWhitney U test and expressed as the median and
interquartile range. Categorical variables were compared between
groups using the chi-squared or Fisher exact test, as appropriate.
Correlations were tested with the Spearman correlation test. The
diagnostic performance of HBsAg and HBcrAg levels was evaluated
by receiver operating characteristic curve analysis. The cut-off
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, IL, USA).
Results
Baseline characteristics
Of the 213 consecutive treatment-naive HBeAg-negative patients evaluated, 202 were included: 135 (66.8%) were classiﬁed as
inactive carriers, 12 (6%) as active carriers, and 55 (27%) as intermediates (see Supplementary material, Fig. S1). 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.
862
M. Riveiro-Barciela et al. / Clinical Microbiology and Infection 23 (2017) 860e867
Table 1
Baseline characteristics according to the stage of HBV infection
Age, years, median (IQR)
Male, n (%)
Genotype, n (%)
A
B or C
D
E
F or H
Mixed A/E
ALT, IU/mL, median (IQR)
Platelets, 10E9/L, median (IQR)
Albumin, mg/dL, median (IQR)
HBsAg, logIU/mL, median (IQR)
HBcrAg, logU/mL, median (IQR)a
HBV DNA, logIU/mL, median (IQR)
Total (n ¼ 202)
Inactive carriers (n ¼ 135)
HBV activity (n ¼ 67)
p value
46 (36e56)
112 (55%)
48 (39e57)
81 (60%)
43 (32e54)
31(47%)
0.004
0.07
75 (37%)
5 (2%)
58 (29%)
36 (18%)
24 (12%)
4 (2%)
18 (13e28)
225 (188e251)
4.4 (4.2e4.6)
3.5 (2.6e4.1)
2.0 (2e2.5)
2.7 (2.1e3.3)
52 (38%)
1 (1%)
38 (28%)
26 (19%)
16 (12%)
2 (2%)
16 (11e24)
222 (185e248)
4 (4.2e4.6)
3.3 (2.4e4.0)
2.0 (2.0e2.0)
2.5 (1.9e2.9)
23 (34%)
4(6%)
20 (30%)
10 (15%)
8 (12%)
2 (3%)
23 (16e33)
227 (197e255)
4.4 (4.1e4.6)
3.7 (3.2e4.1)
2.0 (2.0e3.3)
3.5 (3.0e3.8)
0.98
<0.001
0.25
0.81
0.003
<0.001
<0.001
Abbreviations: ALT, alanine aminotransferase; HBcrAg, hepatitis B core-related antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; IQR, interquartile range.
a
Available in 134 patients.
Serum HBcrAg levels according to the phase of chronic hepatitis B
infection and HBV genotype
Baseline characteristics are summarized in Table 1 and in the
Supplementary material (Table S1). The predominant genotypes
were A (37%), D (29%), E (18%), and F or H (12%). Patients with HBV
activity were younger, and had higher ALT, HBV DNA, HBsAg and
HBcrAg levels than inactive carriers (Table 1). Distribution of HBV
genotypes was similar between inactive carriers and patients with
HBV activity (p 0.98). Overall, mean HBV DNA levels were similar
between the main genotypes (p 0.91). Univariate and multivariate
analyses of baseline characteristics between inactive carriers and
patients with HBV activity are summarized in the Supplementary
material (Table S2).
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) (see Supplementary material, Table 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-infected patients (Fig. 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 in genotype E
patients (r 0.49, p 0.057) (Fig. 2b). All HBV genotype H and F patients had HBcrAg levels <2 logU/mL, the lower limit of detection,
which precluded the study of correlations.
There was a moderate correlation between HBsAg and HBcrAg
levels in patients with genotypes A (r 0.55, p <0.001) and D (r 0.41, p
0.01), and no correlations in genotype E, F or H infection (Fig. 2b).
Serum HBsAg levels according to the phase of chronic hepatitis B
infection and HBV genotype
Overall, HBsAg levels were lower in inactive carriers than in
patients with HBV activity (p 0.003). HBsAg levels differed signiﬁcantly (p <0.001) across the HBV genotypes, being highest for genotype F or H, followed by E, A, and the lowest, D (Table 2). When
HBsAg levels were compared between these two groups according
to HBV genotype, signiﬁcant differences were only observed for
genotypes A and D (p 0.002 and p <0.001, respectively) (Fig. 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 (Fig. 2a).
HBsAg and HBcrAg levels to identify HBV inactive carriers
Overall, the area under the receiver operating characteristic
curve (AUC) for identiﬁcation of inactive carrier status was 0.67
(95% CI 0.56e0.77; p 0.003) for HBcrAg and 0.63 (95% CI 0.55e0.71;
p 0.001) for HBsAg. The cut-off associated with 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
Table 2
Comparison of HBsAg and HBcrAg levels according to HBV genotype and disease activity in HBV infection
Total
Inactive carriers
HBV activity
n
HBsAg, logIU/mL, median (IQR)
HBcrAg, logU/mL, median (IQR)
n (%)
HBsAg, logIU/mL, median (IQR)a
HBcrAg, logU/mL, median (IQR)b
n (%)
HBsAg, logIU/mL, median (IQR)c
HBcrAg, logU/mL, median (IQR)d
Total
GT A
GT D
GT E
GT F or H
p value
202
3.5 (2.6e4.1)
2.0 (2.0e2.5)
135 (67%)
3.3 (2.4e4.0)
2.0 (2.0e2.0)
67 (33%)
3.7 (3.2e4.1)
2.0 (2.0e3.3)
75
3.5 (3.1e4.0)
2.0 (2.0e2.7)
52 (39%)
2.7 (3.3e3.8)
2.0 (2.0e2.2)
23 (34%)
3.8 (3.5e4.2)
2.9 (2.0e3.4)
58
2.7 (2.1e3.5)
2.0 (2.0e2.0)
38 (28%)
2.4 (1.8e2.9)
2.0 (2.0e2.0)
20 (30%)
3.4 (2.9e3.7)
2.0 (2.0e2.6)
36
3.7 (3.0e4.2)
2.0 (2.0e2.5)
26 (19%)
3.7 (2.9e4.2)
2.0 (2.0e2.3)
10 (15%)
3.8 (3.2e4.2)
2.0 (2.3e5.8)
24
4.3 (4.1e4.4)
2.0 (2.0e2.0)
16 (12%)
4.3 (3.9e4.5)
2.0 (2.0e2.0)
8 (12%)
4.3 (4.2e4.4)
2.0 (2.0e2.0)
d
<0.001
0.052
d
<0.001
0.63
d
0.003
0.02
Abbreviations: GT, genotype; HBcrAg, hepatitis B core-related antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; IQR, interquartile range.
a
HBsAg in inactive carriers: p <0.001 for genotype A versus D; p 0.57 for genotype A versus E; p <0.001 for genotype A versus F or H; p <0.001 for genotype D versus E;
p <0.001 for genotype D versus F or H; p 0.013 for genotype E versus F or H.
b
HBcrAg in inactive carriers: p 0.55 for genotype A versus D; p 0.75 for genotype A versus E; p 0.24 for genotype A versus F or H; p 0.52 for genotype D versus E; p 0.48 for
genotype D versus F or H; p 0.14 for genotype E versus F or H.
c
HBsAg in patients with HBV activity: p 0.02 for genotype A versus D; p 0.87 for genotype A versus E; p 0.06 for genotype A versus F or H; p 0.09 for genotype D versus E;
p <0.001 for genotype D versus F or H; p 0.07 for genotype E versus F or H.
d
HBcrAg in HBV activity: p 0.03 for genotype A versus D; p 0.94 for genotype A versus E; p 0.01 for genotype A versus F or H; p 0.34 for genotype D versus E; p 0.21 for
genotype D versus F or H; p 0.13 for genotype E versus F or H.
M. Riveiro-Barciela et al. / Clinical Microbiology and Infection 23 (2017) 860e867
863
Fig. 1. Comparison of mean hepatitis B surface antigen (HBsAg) and hepatitis B core-related antigen (HBcrAg) levels between inactive carriers and patients with hepatitis B virus
(HBV) activity showed statistical differences in patients infected by genotype A (HBsAg and HBcrAg) and D (HBsAg), but not in those with genotypes E, F or H infection.
27.3%). In genotype A, the HBcrAg AUC was higher than that of
HBsAg (AUC 0.80, 95% CI 0.65e0.95; p <0.001, and AUC 0.73, 95% CI
0.60e0.85; p 0.002, respectively). In genotype D, only HBsAg
determination had sufﬁcient discriminatory power to identify
inactive carriers (AUC 0.78, 95% CI 0.66e0.90; p <0.001). Higher
HBsAg levels 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 82.2%; negative predictive
value 50%; diagnostic accuracy 69.3%). However, the previous cutoffs were not useful for proper classiﬁcation of inactive 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).
The diagnostic accuracy of HBcrAg determination was higher
than that of HBsAg for identifying inactive carriers. A single HBV
DNA determination 2000 IU/mL together with HBcrAg 3 logU/
mL yielded a positive predictive value and diagnostic accuracy
>85% for all genotypes except H or F, which showed values of 63%
and 73%, for these two indexes, 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 abovementioned cut-offs (see Supplementary material, Table S4).
HBsAg determination with or without HBV DNA values did not
achieve higher diagnostic accuracy than the combined cut-offs of
HBcrAg 3 logU/mL plus HBV DNA 2000 IU/mL.
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 e0.035 to 0.11; p 0.32; month 12: mean difference 0.069, 95% CI
e0.024 to 0.16; p 0.15). In the comparison of consecutive HBsAg
levels in inactive carriers, 48 patients had at least two determinations separated by 12 months, and a signiﬁcant decay in
HBsAg levels was observed in this group (mean difference 0.16, 95%
CI 0.078e0.23; 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).
Discussion
In this prospective study of well-classiﬁed HBeAg-negative patients infected by HBV genotypes A and D to F, we found considerable variability in HBsAg levels across the 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 genotype A and D patients, which made HBsAg
quantiﬁcation a good biomarker for 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 particularly useful to identify genotype D inactive carriers, with a positive predictive value of 76% and diagnostic accuracy of 74%. These results stem from the low HBsAg 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 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 of 3.7 logIU/mL yielded the best performance for identifying
inactive carriers: positive predictive value 82% and diagnostic accuracy 69%.
HBcrAg is a recently described serum marker formed by three
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
nucleoside analogues [20,21]. Data are scarce on the 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
864
M. Riveiro-Barciela et al. / Clinical Microbiology and Infection 23 (2017) 860e867
Fig. 2. Correlations between hepatitis B virus (HBV) DNA and both hepatitis B surface antigen (HBsAg) (a) and hepatitis B core-related antigen (HBcrAg) (b) reached statistical
signiﬁcance. Independent analysis by each genotype showed that correlations were only present in patients infected by genotype A (HBsAg and HBcrAg) and D (only HBsAg).
Moderate correlations between HBsAg and HBcrAg levels (c) were found in genotype A and D patients.
genotypes than HBsAg quantiﬁcation: a single HBV DNA determination 2000 IU/mL plus HBcrAg levels of 3 logU/mL showed an
overall positive predictive value and diagnostic accuracy of >85%.
There is extensive information concerning HBsAg levels in HBV
genotypes B and C [5,7,22e24]. However, data are scarce for genotypes E to H, the most common genotypes in developing
countries, where HBV is more prevalent [18,19,25,26]. As HBV DNA
monitoring is not currently available worldwide [27], it would be
useful to have a serological marker that can identify inactive carriers in a single determination with less sophisticated technology
and easier manipulation and preservation of serum samples [9].
HBsAg quantiﬁcation could be a good candidate, but the signiﬁcant
M. Riveiro-Barciela et al. / Clinical Microbiology and Infection 23 (2017) 860e867
865
Fig. 2. (continued).
variation in HBsAg 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 128
HBeAg-negative HIVeHBV co-infected patients with HBV genotype
E (mean 3.82 logIU/mL) [28] and in a European cohort of HBV-
infected patients, where higher HBsAg levels 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
Table 3
Diagnostic accuracy and predictive values of HBsAg and HBcrAg cut-offs for identifying inactive carriers status according to HBV genotype
Population
Total
GT A
GT D
GT E
GT F or H
Total
n ¼ 202
55/69
(80%)
53/133
(40%)
108/202
(53%)
GT A
n ¼ 75
16/18
(89%)
21/57
(37%)
37/75
(49%)
GT D
n ¼ 58
29/35
(83%)
14/23
(61%)
43/58
(74%)
GT E
n ¼ 36
8/10
(80%)
8/26
(31%)
16/36
(44%)
GT F or H
n ¼ 24
1/1
(100%)
8/23
(35%)
9/24
(38%)
Total
n ¼ 202
PPV
67/82
(82%)
NPV
52/120
(43%)
Diagnostic accuracy
119/202
(59%)
HBsAg 3.21 logIU/mL + HBV DNA 2000 IU/mL
Population
Total
n ¼ 202
PPV
67/75
(89%)
NPV
59/127
(46%)
Diagnostic accuracy
126/202
(62%)
HBcrAg 3 logU/mL
Population
Total
n ¼ 134
PPV
88/120
(73%)
NPV
12/14
(86%)
Diagnostic accuracy
100/134
(75%)
HBcrAg 3 logU/mL + HBV DNA 2000 IU/mL
Population
Total
n ¼ 134
PPV
88/103
(86%)
NPV
29/31
(91%)
Diagnostic accuracy
117/134
(87%)
GT A
n ¼ 75
24/26
(92%)
21/49
(43%)
45/75
(60%)
GT D
n ¼ 58
31/38
(82%)
13/20
(65%)
44/58
(76%)
GT E
n ¼ 36
10/12
(83%)
8/24
(33%)
18/36
(50%)
GT F or H
n ¼ 24
1/1
(100%)
8/23
(35%)
9/24
(38%)
GT A
n ¼ 75
24/26
(92%)
21/49
(43%)
45/75
(60%)
GT D
n ¼ 58
31/33
(94%)
18/25
(67%)
49/58
(84%)
GT E
n ¼ 36
10/11
(91%)
9/25
(36%)
19/36
(53%)
GT F or H
n ¼ 24
1/1
(100%)
8/23
(35%)
9/24
(38%)
GT A
n ¼ 65
47/57
(82%)
7/8
(88%)
54/65
(83%)
GT D
n ¼ 38
24/35
(69%)
2/3
(67%)
26/38
(68%)
GT E
n ¼ 16
11/14
(79%)
2/2
(100%)
13/16
(81%)
GT F or H
n ¼ 11
5/11
(46%)
0/0
(0%)
5/11
(46%)
GT A
n ¼ 65
47/52
(90%)
12/13
(92%)
59/65
(91%)
GT D
n ¼ 38
24/28
(86%)
9/10
(90%)
33/38
(87%)
GT E
n ¼ 16
11/12
(92%)
4/4
(100%)
15/16
(94%)
GT F or H
n ¼ 11
5/8
(63%)
3/3
(100%)
8/11
(73%)
HBsAg <3 logIU/mL
Population
PPV
NPV
Diagnostic accuracy
HBsAg 3.21 logIU/mL
Population
Abbreviations: GT, genotype; HBcrAg, hepatitis B core-related antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; NPV, negative predictive value; PPV, positive
predictive value.
866
M. Riveiro-Barciela et al. / Clinical Microbiology and Infection 23 (2017) 860e867
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 HBV activity. In a retrospective
multicentre 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 E, F or H. In the present
study, the correlations between HBV DNA and both HBsAg and
HBcrAg are lower than previously reported [30,31]. This may be
because only HBeAg-negative patients were enrolled on our study,
and most were inactive carriers with HBV DNA persistently
2000 IU/mL.
Our study has several limitations, one of them being the heterogeneous distribution of HBV genotypes. This occurred because
patients were unselected and prospectively included, and that
implied a higher percentage of the more prevalent HBV genotypes
in 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 inactive carriers regardless of
HBV genotype should be validated in a larger cohort. One 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.
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 taken into account
when this serum marker is used for clinical purposes.
In conclusion, in a large cohort of well-categorized HBeAgnegative 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.
Funding
This study was funded in part by the Instituto de Salud Carlos III
(grant number PI12/01893) and by Gilead (grant number GLD13/
00137).
Transparency declaration
MB and RE have served as advisors to Gilead, Bristol-Myers
Squibb and Novartis. MB and MRB have received research grants
from Gilead. The other authors have no personal interests to
declare.
Acknowledgements
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. English writing support
was provided by Celine Cavallo.
Contribution to authorship
MB and FRF act as guarantors of this article. MB, RE and MRB
designed the study. MRB, MB and SS selected the patients. MH and
DT performed quantiﬁcation of HBsAg and HBV DNA. RC, MB and
LN carried out HBV DNA ampliﬁcation and genotyping. MRB, MB
and 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.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.cmi.2017.03.003.
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