HBV Testing

Algorithm and
Interpretation
Prof Dr Yasmin A Malik
MPath 2 (Medical Microbiology), UM
9 October 2019
 HBV Structure

• Family: Hepadnaviridae
• 4 serotypes, 10 genotypes (A-J)
• Partially dsDNA enveloped virus (survives outside body for at
least 7 days)
 HBV Genotypes
 The genotypes may play an important role in
 progression of HBV-related liver disease, and
 response to interferon (IFN) therapy
 Genotype A (vs. B-D) is associated with
significantly higher rates of HBeAg & HBsAg loss
with IFN therapy.
 Studies from Asia found that genotype B (vs. C)
is associated with
 HBeAg seroconversion at an earlier age,
 more sustained remission after HBeAg seroconversion,
 less active hepatic necroinflammation,
 a slower rate of progression to cirrhosis, and
 a lower rate of HCC development

Hepatology, 2018
 HBV Prevalence
 In 2015,
 257 million people were living with chronic hepatitis B
infection (defined as HBsAg positive)
 hepatitis B still resulted in around 887,000 deaths, mostly
from cirrhosis and hepatocellular carcinoma. In 2016
 only 10.5% of all people estimated to be living with
hepatitis B) were aware of their infection, and
 16.7% of the people diagnosed were on treatment.
 Global seroprevalence
 WHO Western Pacific Region: 6.2%
 WHO African Region: 6.1%
 WHO Eastern Mediterranean Region: 3.3%
 WHO South East Asian Region: 2.0%
 WHO European Region: 1.0%
 WHO Americas Region: 0.7%
WHO, Jul 2019
 Hepatitis B:
outcome according to age at infection
100 100

Symptomatic infection (%)

80 80
Chronic infection

60 Chronic infection 60
(%)

40 40

20 20
Symptomatic infection
0 0
Birth 1–6 months 7–12 months 1–4 years Older children
and adults
Age at infection

Chronic infection occurs in

• 90% of infants infected at birth,
• 6% of those infected after 5 years of age
CDC, 2007
Serologic Course of HBV Infection
Chronic HBV Infection

WHO, Feb 2017

 Chronic Hepatitis B Diagnosis
Single Assay with HBsAg seroprevalence >0.4%

HBsAg (A1)

HBsAg (A1) + HBsAg (A1) -

(Reactive) (Non-reactive)
Report positive Report negative

Compatible with No evidence of

HBV infection HBV infection

Proceed to NAT testing Advise retesting =/-

for quantification of immunization if still at risk
viraemic infection
WHO, Feb 2017
 Chronic Hepatitis B Diagnosis
Two Assays with HBsAg seroprevalence <0.4%

HBsAg (A1)

HBsAg (A1)+ HBsAg (A2) HBsAg (A1) -

(Non-reactive)
HBsAg (A1) + HBsAg (A1) + Report negative
HBsAg (A2) + HBsAg (A2) -
Report positive Report negative
No evidence of
HBV infection.
Compatible with Inconclusive Advise retesting =/-
HBV infection. result immunization if still at risk
Proceed to NAT testing
for quantification of Further testing
viraemic infection as appropriate
WHO, Feb 2017
 Screening, Counseling
and Prevention of
Hepatitis B
Update on Prevention, Diagnosis, and
treatment of Chronic Hepatitis B:
American Association for the Study of Liver
Disease 2018 Hepatitis B Guidance
 Interpretation of Screening Tests
for HBV Infection
HBsAg AntiHBc AntiHBs Interpretation Management Vaccinate

+ + - HBV infection. Additional testing & No

Chronic HBV if management
pos >6 mths needed

- + + Past HBV No further No

infection management unless
resolved immunocompromised

- + - Past HBV HBV DNA if Yes, if not

infection, immunocompromised from area of
resolved or patient intermediate
false positive or high
endemicity

- - + Immune No further testing

Hepatology, 2018
 Reasons for isolated AntiHBc
1. Among intermediate- to high-risk populations,
 Commonly due to previous exposure to HBV infection;
 Majority of these persons recovered from acute HBV infection
earlier in life and anti-HBs titers have waned to undetectable
levels,
 Some had been chronically infected with HBV for decades
before clearing HBsAg.
2. With new, more specific anti-HBc tests, anti-HBc may be
a false-positive test result, particularly in persons from
low- prevalence areas with no risk factors for HBV
infection
3. Anti-HBc may be the only marker of HBV infection
during the window phase of acute hepaitis B; these
persons should test positive for anti-HBc IgM
4. Reports exist of HBsAg mutations leading to false-
negative HBsAg results

Hepatology, 2018
 Use & Issues of AntiHBc testing
 For blood donors and organ donors (if feasible)
 Routinely performed due to risk of HBV transmission
 Specificity in blood donors: 99.88%
 Specificity in non-HBV medical conditions: 96.85%
 Previously HBV infected, recovered and now
immunocompromised
 At risk for potential reactivation
 Further testing to be done of antiHBc-positive
 AntiHBcIgM - to detect current infection
 AntiHBe – past resolving infection
 HBV DNA: if pos, indicates infectivity but, neg may
mean low HBV DNA

Hepatology, 2018
 Counseling if only AntiHBc positive
1. Anti-HBc is not routinely recommended unless
 HIV infected, or
 about to undergo HCV therapy or immunosuppressive
treatment
2. Persons who are also HBsAg-negative are not at risk
of transmission of HBV, either sexually or to close
personal contacts.
3. If from an area with low endemicity with no risk
factors for HBV, should be given the full series of
hepatitis B vaccine. (Likely non-immune due to false pos anti-HBc)
are
4. If have no high risk behaviour re not recommended
for vaccination unless they are HIV positive or
immunocompromised -> ? refers to high/intermediate endemicity areas

Hepatology, 2018
 Definition and Phases of
Chronic Hepatitis B
infection
Update on Prevention, Diagnosis, and
treatment of Chronic Hepatitis B:
American Association for the Study of Liver
Disease 2018 Hepatitis B Guidance
 Diagnostic Criteria for
Chronic Hepatitis B
1. HBsAg present for ≥ 6 months
2. Serum HBV DNA level varies:
 HBeAg positive CHB: HBV DNA level >20,000 IU/mL
 HBeAg negative CHB: HBV DNA level 2,000-20,000 IU/mL
3. Normal or elevated ALT and/or AST levels
4. Liver biopsy results show
 chronic hepatitis with variable necroinflammation
and/or fibrosis

Hepatology, 2018
 Diagnostic Criteria for
Immune-Tolerant CHB
1. HBsAg present for ≥ 6 months
2. Serum HBV DNA level varies:
 HBeAg positive CHB: HBV DNA level >20,000 IU/mL
 HBeAg negative CHB: HBV DNA level >2,000 IU/mL
3. Intermittently or persistently elevated ALT
and/or AST levels
4. Liver biopsy or noninvasive test results show
 chronic hepatitis with moderate or severe
necroinflammation and/or fibrosis

Hepatology, 2018
 Diagnostic Criteria for Inactive CHB

1. HBsAg present for ≥ 6 months

2. HBeAg negative, anti-HBe positive
3. Serum HBV DNA <2,000 IU/mL
4. Persiostently normal ALT and/or AST levels
5. Liver biopsy confirms
 Confirms absence of significant necroinflammation and
 Show variable levels of fibrosis

Hepatology, 2018
Selected Diagnostic Tests
Used in Management of
Chronic Hepatitis B
Update on Prevention, Diagnosis, and
treatment of Chronic Hepatitis B:
American Association for the Study of Liver
Disease 2018 Hepatitis B Guidance
 Treatment for Chronic Hepatitis B
 Aims of treatment:
 slow the progression of cirrhosis,
 reduce incidence of liver cancer, and
 improve long term survival.
 Only 10%-40% will require treatment.
 WHO recommends tenofovir or entecavir
 rarely lead to drug resistance (unlike the other drugs),
 cheaper (off-patent), simple to take (1 pill a day), and
 have few side effects, so require only limited monitoring
 Unfortunately, in most people,
 treatment only suppresses replication of the virus, thus
 treatment must be continued for life.

WHO, Jul 2019

 Assessment for HBV Treatment
CIRRHOSIS
• Clinical criteria
• Non-invasive tests (LFTs, platelet, Fibroscan)

YES NO

ALL AGES : >30 years (in particular) AGE: <30 years

ALT ALT ALT ALT

Persistently Intermittently Persistently Persistently
abnormal abnormal normal normal

HBV DNA HBV DNA HBV DNA HBV DNA

>20,000 IU/ml 2000- 20,000 <2000 IU/ml <2000 IU/ml
IU/ml

INITIATE THERAPY
AND MONITOR DEFER TREATMENT AND MONITOR

WHO 2017
 Monitoring & Stopping HBV Treatment
Monitor
treatment
Stop

WHO 2017
 HBV-DNA Quantitation
 A crucial component in
 evaluation of patients with CHB, and
 assessment of efficacy of antiviral treatment.
 Method used: real-time PCR
 sensitivity of 5-10 IU/mL and
 dynamic range up to 7 log10 IU/mL
 Some patients with CHB have widely fluctuating
HBV-DNA levels
 Thus, serial monitoring of HBV-DNA levels is more
important than any single arbitrary cut-off value in
determining the need for treatment.
 The 20,000-IU/mL cut-off is an arbitrary value, which
reflects the detection limit of historical non–PCR
assays.
Hepatology, 2018
 Quantitative HBsAg (qHBsAg)
cccDNA= Covalently closed circular DNA

 Quantitative HBsAg (qHBsAg)

 reflects cccDNA and intrahepatic DNA levels, and
 measures HBsAg that arises from integrated DNA,
thereby reducing its specificity as a biomarker for
viral replication.
 qHBsAg levels vary by
 genotype (higher in A), and
 presence of preS/S mutants
 Measured using reproducible, automate &
standardised (IU/mL) assays
 Architect QT assay [Abbott],
 Elecsys HBsAg III Quant [Roche],
 Liaison XL [DiaSorin]

Hepatology, 2018
 Quantitative HBsAg (qHBsAg)
 Levels of HBsAg are generally higher in HBeAg-positive
patients than HBeAg-negative patients
 Inactive CHB HBeAg-neg patients show
 low qHBsAg (<1,000 IU/mL), and
 low HBV DNA (<2,000 IU/mL)
 Helps predict response to therapy
 For peg-IFN treatment of HBeAg-positive patients, qHBsAg
<1,500 IU/mL at week 12 resulted in likelihoods of 57% for
HBeAg seroconversion and 18% for HBsAg loss.
 For NA treatment of HBeAg-negative patients, a >1 log
decline in qHBsAg predicted increased loss of HBsAg, and
qHBsAg level <100 IU/mL were associated with a
sustainable off-treatment response following 3 years or
more of consolidation therapy

Hepatology, 2018
 Quantitative HBsAg (qHBsAg)
(Guidance Statements)
 Essential to guide treatment decisions,
including
 initiation of treatment, and
 evaluation of a patient’s response to
antiviral treatment.
 Can be useful in managing patients
receiving peg-IFN therapy.
 Not recommended for the routine testing or
follow-up of patients with CHB

Hepatology, 2018
 Viral Resistance Testing
 Drug resistance mutations in treatment-naive
patients are rare.
 Defined as a 1-log10 (10-fold) increase in serum HBV
DNA from nadir during treatment in a patient who
had an initial virological response.
 Useful in patients
 with past treatment experience,
 with persistent viremia on NA therapy, or NA= Nucleoside analogue
 who experience virological breakthrough during treatment.
 Methods used:
 restriction fragment length polymorphism (RFLP) analysis,
 hybridization, and
 sequencing.
Hepatology, 2018
Additional Information
 Standard Hepatitis B Vaccine
 2 options for childhood vaccination
 a 3-dose schedule: first dose (monovalent) given at birth and
the second and third doses (monovalent or combined
vaccine) given at the same time as the first and third doses of
DTP vaccine); or
 a 4-dose schedule: monovalent birth dose followed by 3
monovalent or combined vaccine doses, usually given with
other routine infant vaccines.
 Complete series induces protective antibody levels in
>95% of infants, children & young adults.
 Protection lasts at least 20 years & may be lifelong.
 Thus, WHO does not recommend booster vaccinations for
persons who have completed the 3 dose vaccination
schedule.

WHO Jul 2019

 HBV Vaccine with aluminum adjuvant

Current Types of scheduling

Standard schedule (e.g. MSD, GSK)
IM in a 3-dose series at 0, 1, and 6 months
Accelerated scheduling
IM in a 3-dose series at 0, 1, and 2 months
IM in a 3-dose series at 0, 7, and 21 days (20 years
old & older)
4th dose needs to be given at 12 months (as titre
after 3rd dose is lower than after the standard 0,1,6
schedule)
 Hepatitis B Vaccine with novel adjuvant
 On November 9, 2017, Heplisav-B (HepB-CpG), a
single-antigen HepB recombinant vaccine with a
novel immunostimulatory sequence adjuvant,
was approved by the US FDA for the prevention
of HBV in persons aged ≥18 years.
 Schedule and Mode of administration
 IM, 2 doses, 1 month apart.
 On February 21, 2018, the Advisory Committee on
Immunization Practices (ACIP) recommended
HepB-CpG for use in persons aged ≥18 years.

MMWR, April 2018;67(15): 455-58

 HEPLISAV-B

 Each 0.5-mL dose is formulated to contain

 20 mg of yeast-derived recombinant HBsAg and
 3000 mg of CpG 1018 adjuvant.
 It is formulated without preservatives
 Seroprotective antiHBs levels (≥10 mIU/mL )
 Engerix B (among 3214 subjects) : 70.5-90.2%
 HEPLISAV-B (among 7056 subjects) : 90-100%
 Not available in Malaysia

MMWR, April 2018;67(15): 455-58

 Trivalent HBV Vaccine
 Contains pre-S1, pre-S2, and S antigens of
hepatitis B
 15 months comparative study initiated in
December 2017 (given IM on days 0,1,6 months):
 10 mg dose of trivalent vaccine (Sci-B-Vac, VBI Vaccines)
 20 mg dose of monovalent vaccine (Engerix-B, GSK)
 Results*:
Higher rates of protection among adults >18 years, &
acceptable safety profile

*Langley JM et al. Presented at ID Week 2-6 October 2019, Washington, USA

 Latest Influenza vaccine composition
Season Type A (H1N1) Type A (H3N2) Type B
2018-2019 A/Michigan/45/20 A/Singapore/INFIMH-16- B/Colorado/06/2017
Northern 15 (H1N1)pdm09 0019/2016 (Victoria/2/87 lineage)
Hemisphere
(Mac 2018) Quadrivalent Vaccine: B/Phuket/3073/2013 (Yamagata/16/88 lineage)
2019 A/Michigan/45/20 B/Colorado/06/2017
Southern A/Switzerland/8060/2017
15 (H1N1)pdm09 (Victoria/2/87 lineage)
Hemisphere
(Sept 2018) Quadrivalent Vaccine: B/Phuket/3073/2013 (Yamagata/16/88 lineage)

2019-2020 A/Brisbane/02/20 B/Colorado/06/2017

Northern A/Kansas/14/2017 (H3N2)
18 (H1N1)pdm09 (Victoria/2/87 lineage)
Hemisphere
(Mac 2019) Quadrivalent Vaccine: B/Phuket/3073/2013 (Yamagata/16/88 lineage)
2020 A/Brisbane/02/20 A/South Australia/34/2019 B/Washington/02/2019
Southern 18 (H1N1)pdm09 (H3N2) (Victoria/2/87 lineage)
Hemisphere
(Sept 2019) Quadrivalent Vaccine: B/Phuket/3073/2013 (Yamagata/16/88 lineage)
WHO, Sept 2019