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Annex 5.10
PICO 8 - Diagnostic accuracy of HBsAg/HBeAg test versus NAT to confirm successful treatment response: a meta-analysis and review of the
literature
London School of Hygiene and Tropical Medicine team
*Olivia Varsaneux, *Ali Amini, Weiming Tang, Wen Chen, Debi Boeras, Jane Falconer, Helen Kelly,
Joseph Tucker, Rosanna Peeling (Team lead)
London School of Hygiene and Tropical Medicine team
*Co-leaders of this Review
September 2015
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1. Executive summary
Background: Advances in hepatitis B virus detection technology create new opportunities for
enhancing screening, referral and treatment. The purpose of this review was to determine the
diagnostic accuracy of HBsAg or HBeAg test versus nucleic acid testing (NAT) to confirm successful
treatment response among patients receiving treatment for HBV.
Method: A literature search was conducted focused on hepatitis B, diagnostic tests and diagnostic
accuracy. Studies were included if they evaluated an assay to determine the sensitivity and
specificity of a HBsAg or HBeAg test compared to a quantitative HBV RNA reference among
humans. Two reviewers performed a quality assessment of the studies and extracted data for
estimating test accuracy.
Results: It was found that, despite HBV NAT being considered the gold standard in confirming
response to treatment, both HBsAg and HBeAg were useful in monitoring patients receiving
treatment as in many resource-limiting settings NAT is not readily available. Studies showed that
the kinetics of HBsAg and HBV DNA followed similar profiles during treatment with pegylated
interferon (PEG-IFN) and follow up in patients who developed sustained virological response
(SVR). Further studies determined that this correlation was present for all four genotypes. It was
also reported that HBsAg quantification can allow for detection of active cases of chronic HBV
from true inactive carriers, therefore reducing the need to rigorously monitor HBV DNA levels.
Studies showed that HBeAg was capable of differentiating late responders from non-responders
to HBV DNA after 24 weeks of treatment.
Conclusions: There is limited evidence for the sole use of HBsAg or HBeAg compared to HBV DNA
for monitoring treatment response. More studies are needed to determine which tests for HBV
antigen detection may be useful as a marker of treatment response for which therapeutic agent.
2.Background
An estimated 240 million individuals worldwide1 are chronically infected with hepatitis B virus
(HBV) and there are an estimated 4 million acute HBV infections each year. Of those with chronic
hepatitis B infection, 20–30% will develop cirrhosis2 or hepatocellular carcinoma,3 leading to
approximately 650 000 deaths each year.4 However, most individuals with chronic HBV infection
are not aware of their serostatus, contributing to delayed diagnosis and complications from
advanced disease.5 HBV testing is critically important in order to refer infected individuals to HBV
treatment and care, to refer uninfected individuals to vaccination and to mobilize prevention and
control efforts.
The introduction of NAT is an integral step in the control of the disease as it allows for
rapid diagnosis and early treatment of HBV. The virus can be transmitted by blood from
asymptomatic donors with acute HBV infection before the development of HBsAg or an anti-HBc
response. Therefore, NATs can used to detect HBV DNA in a donor’s blood before antigen or
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antibody response are detected.6 Though NAT testing has been proven to be more sensitive in
detecting viral infections, serological testing is better suited for the detection of active infections.7
Treatment with tenofovir or entecavir is effective for HBV. Their efficacy can be measured
by a sustained reduction in viral load, but the quantitative HBsAg response may remain high. The
data for measuring HBsAg quantitatively largely works for interferon-based agents. Locarini
reviewed the literature on quantitative HBsAg in hepatology and highlighted some of the
challenges of quantitative HBsAg testing in using other therapeutic agents.
In March 2015, the World Health Organization (WHO) published the first guidelines for
the prevention, care and treatment of individuals with chronic HBV infection.5 These guidelines
focused on assessment for treatment eligibility, initiation of first-line therapies, switching and
monitoring. These initial guidelines did not include screening recommendations. Given the large
burden of HBV in low- and middle-income settings where there are limited or no existing HBV
testing guidelines, there is a substantial need for HBV testing guidelines.
Advances in HBV detection technology create new opportunities for enhancing screening,
referral and treatment. Previous systematic reviews on hepatitis B infection have focused on
immunological responses,7 surveillance of cirrhosis8 and treatment.9 Existing systematic reviews10‒
13 on hepatitis B testing focused on point-of-care (POC) tests and included tests with unclear
reference standards. No systematic reviews have examined the diagnostic accuracy of using
HBsAg/HBeAg compared to HBV DNA detection to monitor treatment response.
PICO 8
Among patients receiving treatment for HBV, what is the diagnostic accuracy of HBsAg/HBeAg
test versus NAT to confirm successful treatment response?
P Patients receiving treatment for HBV
I HBsAg/HBeAg testing
C NAT for HBV DNA detection
O Diagnostic accuracy
True negatives (TNs) – who are screen negative and have cleared the HBV infection.
False negatives (FN) – who are screen negative but have HBV infection. These will be
misclassified and treatment will be stopped resulting in disease progression leading to liver-
related morbidity (fibrosis, cirrhosis, end-stage liver disease, hepatocellular carcinoma),
progression of liver disease and mortality.
True positives (TP) – who are screen positive and truly have HBV infection. This will increase
the number of treated cases and cure rate.
False positives (FP) – who are screen positive, but do not have HBV infection. (These will
continue treatment inappropriately, and and will have unnecessary referral).
Costs – cost of testing strategy, including lab reagents and running costs, cost of further
evaluation of a false positive.
Cost–effectiveness
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Acceptability to health-care workers and patients
Other outcomes (missed cases of liver disease because of false-negative results, unnecessary
referral, investigations and/or treatment in false positives).
3. Objectives
The purpose of this review was to identify evidence on the sensitivity and specificity of
HBsAg/HBeAg compared to HBV DNA detection for HBV treatment monitoring and to summarize
the key test characteristics associated with detection of HBsAg/HBeAg.
4. Methodology
We followed standard guidelines and methods for systematic review and meta-analyses of
diagnostic tests.14,15 We prepared a protocol for the literature search, article selection, data
extraction and assessment of methodological quality.
Selection criteria
i. Types of studies
We included observational studies and randomized controlled trials (RCTs) that provide original
data from patient specimens, including cross-sectional and case–control studies, and studied
HBsAg/HBeAg testing compared to a reference standard of HBV DNA detection.
ii. Participants
Little information on participants was provided in the selection of papers included in the
systematic review; therefore, we set a wide inclusion criterion. We included patients of all age
groups from all settings and countries as well as all types of specimens.
iii. Index tests
Studies that utilized commercially available HBsAg/HBeAg and HBV DNA assays were eligible for
inclusion. The following four are the index tests included:
Architect HBsAg assays, Abbott
COBAS AMPLICOR TM HBV Test v2.0 assay. Roche Diagnostics Systems
IMx HBeAg assay, Abbott
Iprobe, Abbott
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iv. Reference standard
The reference standards acceptable for a definitive diagnosis included tests for detection of HBV
by the following HBV DNA detection techniques—polymerase chain reaction (PCR), branched-
chain DNA (bDNA), or transcription-mediated amplification (TMA) and DNA hybridization assays.
Outcome measures
Sensitivity refers to the proportion of samples with true HBV infection diagnosed with positive
HBsAg/HBeAg test confirmed with a positive HBV DNA detection method.
Specificity refers to the proportion of samples with negative HBsAg/HBeAg test confirmed
with a negative HBV DNA detection method.
Search methods
A database search of LILACS, MEDLINE, EMBASE, PubMed, Scopus, Web of Science, Cochrane and
WHO Global Index Medicus was performed through April 2015. No language restriction was
applied. The references of published articles found in the above databases were searched for
additional pertinent materials.
Study selection proceeded in three stages. First, titles/abstracts were screened by a single
reviewer according to standard inclusion and exclusion criteria. Second, full manuscripts were
obtained and assessed against inclusion criteria. Papers were accepted or rejected and reasons
for rejection were specified. Third, two independent reviewers assessed each manuscript and
differences were resolved by a third independent reviewer.
Data extraction
Information on the following variables were extracted by a reviewer if the study met the exclusion
and inclusion criteria—first author, total sample size, country (and city) of sampling, sample type
(oral fluid, finger-prick, venous blood, etc.), point-of-care (Y/N), eligibility criteria, reference
standard, manufacturer, raw cell numbers (true positives, false negatives, false positives, true
negatives), sources of funding and reported conflicts of interest. We define point of care as being
able to give a result within 60 min and having the results guide clinical management at the same
encounter.
Assessment of methodological quality
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Study quality was evaluated using the QUADAS-2 tool,16 the STARD checklist17 and the GRADE
method.18 QUADAS includes domains to evaluate bias in the following categories—risk of bias
(patient selection, index test, reference standard, flow and timing); applicability concerns (patient
selection, index test, reference standard). The GRADE method evaluates the strength of evidence
by assessing the risk and probability of bias, imprecision and inconsistency as well as dose–
respondent gradient and residual confounding.18
5. Results
PRISMA flowchart
Fig. 1. PRISMA flow diagram outlining study selection examining diagnostic accuracy of HBV
antibody tests compared to HBV DNA in confirming successful treatment response
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Characteristics of included studies
Only two of the studies analysed met the PICO criteria and data were extracted from both these
studies. These studies took place in Sweden and the United States of America. The patient
population for the Larsson 2013 study was derived from a clinical setting; there was no
information on the patient population for the Perrillo 1993 study. The assays evaluated for this
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systematic review were Architect HBsAg assays, COBAS AMPLICOR TM HBV Test v2.0 assay, IMx
HBeAg assay and Iprobe. Of these two studies, only one reported sensitivity/specificity of HBsAg
and one reported sensitivity/specificity of HBeAg.
The lack of information on these diagnostic accuracy measures is a large limitation in the quality
of the studies. Other issues with the quality of these studies were insufficient information on the
populations studied, randomization and sample collection.
Table 1. Description of study design, study population and setting of all studies (n=2)
Larsson et al. (2013) monitored HBsAg levels (Architect assays, Abbott) and HBV DNA quantitation
(COBAS AMPLICORTM HBV monitor, Roche) in 160 patients treated for chronic HBV infection at the
Infectious Disease Clinic at Sahlgrenska University Hospital between 1993 and 1995. Sensitivity of
HBsAg compared to HBV DNA was 34% and the specificity was 89%. A correlation between HBsAg
and HBV DNA in serum samples (R2 = 0.39; P< 0.0001) was also noted, in that a 90% reduction of
HBV DNA corresponded to a 48% decline in HBsAg. The authors also measured HBeAg levels and
found that HBeAg-positive patients had a 300 times higher HBV DNA/HBsAg ratio compared to
those who were HBeAg-negative. These results indicate that HBsAg quantification could be
complementary to HBV DNA quantification for treatment monitoring and confirming successful
treatment response.
Perrillo et al. (1993) evaluated whether the HBeAg assay (Abbott IMX) was capable of
providing comparable information to HBV DNA assays (Iprobe, Abbott) during and after IFN
therapy in 29 consecutive, IFN-treated patients and five untreated controls. The authors found
that decremental and incremental changes in HBeAg concentration during and after therapy
mirrored those observed with HBV DNA with a significant correlation (R = 0.768 P>0.0001). Only
56% of HBV DNA-negative patients tested positive for HBeAg but 95% of HBV DNA-positive
samples were also positive for HBeAg. Though this information allows us to understand that
HBeAg concentrations can provide similar clinically relevant information compared to HBV DNA
First
author
Sample
type & size
Country Treatment Study
population
Eligibility
criteria
Index
diagnostic
test
Reference
test
Sensitivity
Specificity
1 Larrson,
2013
Liver tissue
and blood
sample
N= 160
Sweden INF Infectious
Disease clinic
N =160
Patients
with
chronic
HBV
Architect
HBsAg
assays,
Abbott
COBAS
AMPLICORT
M HBV Test
v2.0 assay.
Roche
34% 89%
3 Perrillo,
1993
Plasma
N= 34
United
States of
America
INF ? 29 patients
on
treatment
and 5 neg.
controls
IMx
HBeAg
assay,
Abbott
Iprobe,
Abbott
95% 44%
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assays, it is difficult to state the accuracy of these tests against each other as they are traditionally
used to measure different indicators.
Narrative summary of each systematic review’s findings
Monitoring response to treatment is an essential mechanism in the control of HBV and requires
both the sustained disappearance of HBV DNA and the clearance of HBsAg/ HBeAg from the
blood. The systematic review showed that monitoring of HBeAg concentration can provide
clinically relevant information, though only two of the 6464 studies identified for screening
(Larrson et al. 2013 and Perrillo et al. 1993) were included in the systematic review as they were
the sole articles that met both the inclusion and exclusion criteria for PICO 8 (showed sensitivity
and specificity of assays).
i. Diagnostic accuracy of nucleic acid testing
HBV DNA is essential when determining the presence of the virus as it is quantitatively expressed
and allows for prompt detection of HBV. With the advent of reverse transcriptase-polymerase
chain reaction (RT-PCR), it quickly became regarded as the gold standard or confirming response
to therapy due to its accuracy and cost–effectiveness. However, in many resource-limited
settings, such assays are not widely available, therefore it is important to determine if HBsAg or
HBeAg can be used for monitoring response to treatment.19
ii. Diagnostic accuracy of HBsAg test compared to HBV DNA
Although they did not include specific accuracy values, three supplemental studies provided
useful information on the quantitation of HBsAg for treatment monitoring in chronic HBV
patients. Martinot-Peignoux et al. (2015) reported that the kinetics of HBsAg and HBV DNA
followed similar profiles during treatment with PEG-IFN and follow up in patients who developed
SVR (solid line) (see Fig. 2).18
Fig. 2. Serum HBV DNA and HBsAg kinetics during treatment with PEG-IFN and follow up in
patients who developed SVR (solid line) 20
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This was confirmed by Ganji et al. (2011) who that showed HBsAg had strong correlation with HBV
DNA (r =0.69; P<0.01) for both genotypes investigated.20 Larsson et al. 2014 further proved that
that there was a correlation between HBsAg and HBV DNA for all four genotypes (Fig. 3).19 This
highlights the potential for HBsAg to be a useful serological marker to predict response to
treatment.
Fig. 3 (A–D). Correlation between HBsAg and HBV DNA in genotypes A–D
Fig. 3 (E). Box plot of HBsAg levels in HBeAg-positive and -negative patients by genotype (no
significant differences)19
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Another important use for HBsAg assays is in monitoring treatment response for HBeAg-negative
chronic patients with low HBV DNA levels. Sonneveld et al. (2011) reported that when monitoring
PEG-IFN treatment in patients with chronic hepatitis B, HBsAg reduction is most pronounced in
patients who achieve a response to therapy at 6 months post treatment.19 This suggests that
HBsAg quantification can allow for detection of active cases of chronic HBV from true inactive
carriers, thereby reducing the need to rigorously monitor HBV DNA levels.
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Fig. 4. Hepatitis B surface antigen decline during PEG-IFN treatment of hepatitis B e antigen
(HBeAg)-positive and HBeAg-negative patients26
iii. 4.5.3 Diagnostic accuracy of HBeAg test compared to
HBV DNA
Monitoring HBeAg has been shown to be important due to its association with the disappearance
of replicative viral intermediates and its persistence in the blood once HBV DNA has cleared. 19
Using PEG-IFN alfa-2a, Fried et al. (2008) showed that HBeAg levels proved to be a stronger
indicator of non-response compared to HBV DNA after 24 weeks of treatment. Lower levels of
HBV DNA were seen to closely predict seroconversion (Table 2).
Table 2. Serum HBV DNA at weeks 12 and 24 of treatment: relationship to HBeAg seroconversion
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It was also shown that those who reached HBeAg seroconversion had a consistent decline in their
levels of HBeAg and remained at the lowest levels while under the follow-up period. This was in
contrast to those who failed to achieve seroconversion after treatment was discontinued, as a
rebound was observed allowing for better determining of seroconversion and a higher negative
predictive value (Fig. 5). This highlights the importance of HBeAg in differentiating late responders
from non-responders and is an important aspect of treatment.22
Fig. 5. HBV DNA levels: responders versus non-responders at 24 weeks post treatment – HBeAg
seroconversion 23
However, monitoring treatment response using HBeAg can be complicated as the response may
vary with the therapy used. Non-interferon agents rarely cause HBeAg loss or might cause only a
transient HBeAg loss while on therapy. Interferon agents are toxic and might convert ~35% of
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HBeAg positives to negatives but only in a subset of people with high alanine aminotransferase
(ALT).23
Monitoring HBV treatment response remains a challenge. Guidelines for chronic HBV
management and treatment state that the ideal end-point of treatment should be dictated by a
lack of detectable HBsAg. The use of HBsAg/HBeAg as a marker to detect sustained virological
response is essential, because on-treatment decrease in HBV DNA shows similar patterns for both
sustained responders and relapsers (Fig. 3).20 Due to the infrequency of obtaining this point with
the current anti-HBV agents, the primary goal of antiviral therapy is defined as viral remission,
PCR non-detectability (<300 copies/mL [57 IU/mL]).19,21
For the time being, NAT can be used as the reference standard to confirm this response to
therapy. More studies are needed to determine which tests for HBV antigen detection may be
useful as effective markers of treatment response for therapeutic agents.
References
A. Reference list of studies that met criteria for inclusion in the analysis
1. Larsson R, Eilard A, Malmström S, Hannoun C, Dhillon AP, Norkrans G, et al. HBsAg quantification for
identification of liver disease in chronic hepatitis B virus carriers. Liver Int. 2014;34(7):238–45.
2. Perrillo R, Mimms L, Schechtman K, Robbins D, Campbell C. Monitoring of antiviral therapy with
quantitative evaluation of HBeAg: a comparison with HBV DNA testing. Hepatology. 1993;18(6):1306–
12.
B. Reference list from background
1. Ott JJ, Stevens GA, Groeger J, Wiersma ST. Global epidemiology of hepatitis B virus infection: new
estimates of age-specific HBsAg seroprevalence and endemicity. Vaccine. 2012;30(12):2212–19.
2. Ganem D, Prince AM. Hepatitis B virus infection – natural history and clinical consequences. N Engl J
Med. 2004;350(11):1118–29.
3. Fattovich G, Stroffolini T, Zagni I, Donato F. Hepatocellular carcinoma in cirrhosis: incidence and risk
factors. Gastroenterology. 2004;127(5 Suppl 1):S35–S50.
4. Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V, et al. Global and regional mortality from
235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of
Disease Study 2010. Lancet. 2012;380(9859):2095–128.
5. Guidelines for the Prevention, Care, and Treatment of Persons with Chronic Hepatitis B Infection.
Geneva: World Health Organization; 2015
(http://apps.who.int/iris/bitstream/10665/154590/1/9789241549059_eng.pdf?ua=1&ua=1, accessed
06 June 2016).
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HBV infection in blood donors. N Engl J Med. 2011;364(3):236–47.
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randomized trials]. [Article in Chinese]. Zhong Xi Yi Jie He Xue Bao. 2010;8(4):312–20.
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specificity of hepatitis B surface antigen rapid tests]. [Article in Korean]. Korean J Lab Med.
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hepatitis B infection: a meta-analysis of diagnostic accuracy (1980–2010). Am J Gastroenterol.
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surface antigen – systematic review and meta-analysis. J Clin Exp Hepatol. 2014;4(3):226–40.
14. CRD’s guidance for undertaking reviews in health care. York: Centre for Reviews and Dissemination;
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15. Pai M, Mcculloch M, Gorman, JD, PaiN, Enanoria W, Kennedy G, et al. Systematic reviews and meta-
analyses: an illustrated, step-by-step guide. Natl Med J India. 2004;17(2):86–95.
16. Whiting PF, Rutjes AW, Westwood ME, Mallet S, Deeks JJ, Reitsma JB, et al. QUADAS-2: a revised tool
for the quality assessment of diagnostic accuracy studies. Ann Intern Med. 2011;155(8):529–36.
17. Bossuyt PM, Reitsma JB, Bruns DE, Gatsonis CA, Glasziu PP, Irwig LM, et al. The STARD statement for
reporting studies of diagnostic accuracy: explanation and elaboration. 2003;138(1):W1–W12.
18. Larsson SB, Eilard A, Malmström S, Hannoun C, Dhillon AP, Norkrans G, et al. HBsAg quantification for
identification of liver disease in chronic hepatitis B virus carriers. Liver Int. 2014;34(7):e238–e45.
19. Perrillo R, Mimms L, Schechtman K, Robbins D, Campbell C. Monitoring of antiviral therapy with
quantitative evaluation of HBeAg: a comparison with HBV DNA testing. Hepatology. 1993;18(6):1306–
12.
20. Ganji A, Esmaeilzadeh A, Ghafarzadegan K, Helalat H, Rafatpanah H, Mokhtarifar A. Correlation
between HBsAg quantitative assay results and HBV DNA levels in chronic HBV. Hepat Mon.
2011;11(5):342–5.
21. Martinot-Peignoux M, Asselah T, Marcellin P. HBsAg quantification to optimize treatment monitoring in
chronic hepatitis B patients. Liver Int. 2015;35(1):82–90.
22. Fried MW, Piratvisuth T, Lau GK, Marcellin P, Chow WC, Cooksley G, et al. HBeAg and hepatitis B virus
DNA as outcome predictors during therapy with peginterferon alfa-2a for HBeAg-positive chronic
hepatitis B. Heptaology. 2008;47(2):428–34.
23. Viganò M, Lampertico P. ClinicaliImplications of HBsAg quantification in patients with chronic hepatitis
B. Saudi J Gastroenterol. 2012;18(2):81–6.