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

Page | 538

1. Executive summary

Background: Advances in 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 , 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 (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

Page | 539

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

Page | 540

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

Page | 541

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

Page | 542

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

Page | 543

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

Page | 544

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)

First Sample Country Treatment Study Eligibility Index Reference Sensitivity Specificity author type & size population criteria diagnostic test test

1 Larrson, Liver tissue Sweden INF Infectious Patients Architect COBAS 34% 89% 2013 and blood Disease clinic with HBsAg AMPLICORT sample N =160 chronic assays, M HBV Test N= 160 HBV Abbott v2.0 assay. Roche

3 Perrillo, Plasma United INF ? 29 patients IMx Iprobe, 95% 44% 1993 N= 34 States of on HBeAg Abbott America treatment assay, and 5 neg. Abbott controls

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

Page | 545

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 -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

Page | 546

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

Page | 547

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.

Page | 548

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

Page | 549

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

Page | 550

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.

Page | 551

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– 62. 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.

Page | 552