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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).

6. Krajden M, McNabb G, Petric M. The laboratory diagnosis of hepatitis B virus. Can J Infect Dis Med

Microbiol. 2005;16(2):65–72.

7. Stramer SL, Wend U, Candotti D, Foster GA, Hollinger FB, Dodd RY, et al. Nucleic acid testing to detect

HBV infection in blood donors. N Engl J Med. 2011;364(3):236–47.

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8. Fabrizi F, Martin P, Dixit V, Bunnapradist S, Dulai G. Meta-analysis: the effect of age on immunological

response to hepatitis B vaccine in end-stage renal disease. Aliment Pharmacol Ther. 2004;20(10):1053–

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9. Thompson Coon J, Rogers G, Hewson P, Wright D, Anderson R, Cramp M, et al. Surveillance of cirrhosis

for hepatocellular carcinoma: systematic review and economic analysis. Health Technol Assess.

2007;11(34):1–206.

10. Qin XK, Li P, Han M, Liu JP. [Xiaochaihu Tang for treatment of chronic hepatitis B: a systematic review of

randomized trials]. [Article in Chinese]. Zhong Xi Yi Jie He Xue Bao. 2010;8(4):312–20.

11. Hwang SH, Oh HB, Choi SE,Kim HH,Chang CL,Lee EY, et al. [Meta-analysis for the pooled sensitivity and

specificity of hepatitis B surface antigen rapid tests]. [Article in Korean]. Korean J Lab Med.

2008;28(2):160–8.

12. Shivkumar S, Peeling R, Jafari Y, Joseph L, Pai NP. Rapid point-of-care first-line screening tests for

hepatitis B infection: a meta-analysis of diagnostic accuracy (1980–2010). Am J Gastroenterol.

2012;107(9):1306–13.

13. Khuroo MS, Khuroo NS, Khuroo MS. Accuracy of rapid point-of-care diagnostic tests for hepatitis B

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;

2008.

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.